Treatment of immunological disorders using anti-dc30 antibodies

ABSTRACT

The present invention relates to methods for the treatment of immunological disorders other than cancer, comprising administering proteins characterized by their ability to bind to CD30 and exert a cytostatic or cytotoxic effect on an activated lymphocyte. Such proteins include monoclonal antibodies AC10 and IleFi1. AC10 and HeFi-1 derivatives, and antibodies that compete with AC10 and HeFi-1 for binding to CD30. Other such proteins include multivalent anti-CD30 antibodies and anti-CD30 antibodies conjugated to cytotoxic agents. Treatment modalities with antibodies of the invention are also provided.

This application claims benefit under 35 U.S.C. § 119(e) of U.S.provisional application no. 60/331,750, filed Nov. 20, 2001, which isincorporated by reference herein in its entirety.

1. FIELD OF THE INVENTION

This invention relates to methods and compositions for inducing celldeath or stasis in activated lymphocytes using CD30-binding proteins,and applications for these methods and compositions for the treatment ofimmunological diseases such as autoimmunity, allergy, chronicinflamatory reactions, and graft versus host disease (GVHD). The presentinvention further relates to methods and compositions for treatment ofimmunological disorders by eliminating CD30-expressing lymphocytes usingconjugates of CD30-binding proteins and cytotoxic agents. ExemplaryCD30-binding proteins that are useful in the methods and compositions ofthe present invention include the anti-CD30 antibodies AC10 and HeFi-1and conjugates of AC10 or HeFi-1 and cytotoxic agents.

2. BACKGROUND OF THE INVENTION

2.1 Lymphocytes

Subsequent to antigenic stimulation and cellular expansion, naïve Tcells can develop into phenotypically distinct effector cells. EffectorT cells (helper or cytotoxic T cells) that secrete pro-inflamatorycytokines like IFNγ and lymphotoxin are collectively designated as Th₁or Tc₁ cells, and effector T cells that secrete IL-4, IL-5, IL-6, IL-9,IL-10, and IL-13 are known as Th₂ or Tc₂. Th₁/Tc₁ cytokines inducecell-mediated responses including the activation of CTL andmonocyte/macrophages, whereas Th₂/Tc₂ cytokines induce humoral immuneresponses by enhancing antibody production by B cells. As a result,uncontrolled Th₁/Tc₁ responses are usually manifested as organ-specificautoimmune responses such as rheumatoid arthritis and diabetes. On theother hand, uncontrolled Th₂/Tc₂ responses are usually associated withallergic reactions and systemic autoimmune diseases like systemic lupuserythematosus. The two polarized T cell subsets can modulate each othersactivity in a reciprocal fashion—the presence of Th₂/Tc₂ cytokines canpartially alleviate symptoms resulted from cell-mediated autoimmunityand vice versa (Seder and Mosmann, 1999, in ‘Fundamental Immunology’,4^(th) Ed., pp 879-908; O'Gara and Arai, 2000, Trends Cell Biol.10:542-550).

2.2 CD30

The leukocyte activation marker CD30 is a 105-120 kDa integral membraneglycoprotein and a member of the tumor necrosis factor receptor (TNF-R)superfamily. This family of key immunoregulatory molecules includesCD27, CD40, CD95, 0×40, TNF-R1 and TNF-R2. Originally identified onReed-Sternberg cells in Hodgkin's disease (HD) using the Ki-1 monoclonalantibody (mAb) (Stein et al., 1985, Blood, 66, 848-858; Laudewitz et al,1986, J. Invest. Dermatol. 86:350-354), CD30 has subsequently been foundon anaplastic large cell lymphoma (ALCL), subsets of non-Hodgkin'slymphomas (NHL), as well as in rare solid tumors such as embryonalcarcinomas and seminomas (Chiarle et al., 1999, Clin. Immunol.90:157-164). Under normal conditions expression of CD30 is restricted toactivated T and B cells and absent from resting lymphocytes, restingmonocytes and from normal cells outside of the immune system. CD30 isexpressed at high levels by activated cells in autoimmune disease, andshed CD30 from these cells is detectable in the circulation of patientssuffering from diseases including rheumatoid arthritis (Gerli et al.,2000, J. Immunol. 164, 4399-4407), multiple sclerosis (McMillan et al.2000, Acta Neurol. Scand. 101 :239-243) and systemic sclerosis (Ihn etal., J. Rheumatol. 27:698-702). Histological examination for CD30expression identifies rare, large lymphoid cells in sections of lymphnode, tonsil, thymus, and decidual endometrial cells at the placentalinterface (Durkop et al., 2000, J. Pathol. 190:613-618). CD30 istransiently expressed on T cells in culture after mitogen activation orantigen receptor crosslinking (Horle and Watanabe, 1998, Sem. Immunol.10:457-470), and is constitutively expressed following some viralinfections, e.g., HIV (Romagnani et al., 1996, Immunol. Lett. 51:83-88).

A ligand for CD30 (CD30L) has been identified. CD30L is a type IItransmembrane protein, and it shares sequence and structural homologieswith members of the TNF superfamily (Smith et al., 1993, Cell, 73′.1349-1360). It is believed that CD30L, like TNFα, also exists as atrimer, and CD30L-CD30 interaction induces trimerization of CD30 toinitiate signal transduction to CD30⁺ cells. Expression of CD30L hasbeen demonstrated in activated T cells, neutrophils, eosinophils,resting B cells, epithelial cells and Hassal's corpuscles of the thyimicmedulla, and some leukemic cells (Younes et al. Br. J. Haematol.,93:569-571: Pinto et al., 1996, Blood. 88:3'299-3305: Grüss et al.,1996, Am. J. Pathol. 149, 469-481; Gattei et al., 1997, Blood, 89,2048-2059; Romagnani et al. 1998. Blood 91:3323-3332; Wiley et al.,1996. J. Immunol., 157:3635-3639).

The lymphocyte surface antigen CD30 is an activation marker transientlydisplayed on activated B and T cells and constitutively expressed onsome malignant hematologic cells and on chronically activated-cells inseveral autoimmune diseases. Its role in lymphocyte regulation isbelieved to be one of attenuation, as lack of CD30 in knock-out animalsresults in hyperresponsiveness to immune stimuli, whereas overexpression of the transgene in the thymus results in increased thymocytedepletion. Thus, elimination or attenuation of activated lymphocytes viaCD30-targeted therapy could be efficacious in controlling autoimmune andchronic inflammatory diseases.

The complexity of CD30 in regulating lymphocyte survival has lead todisparate accounts of its effects in model systems. Duckett and Thompson(1997, Genes Dev. 11:2810-21) have shown the initial effect of CD30signaling is to promote survival by recruitment of TRAF1 and TRAF2. Thissignal transduction complex promotes growth and survival-gene expressionvia NF-κB. Following this activation, TRAFs complexed with CD30 are thendegraded, compromising the ability of the cells to further activateNF-κB. Consequently, the cells become sensitized to apoptosis induced byligation of TNF-R1 (Duckett and Thompson, 1997, Genes Dev. 11:2810-21;Arch et al., 2000, Biochem. Biophys. Res. Commun. 272:936-945).Sensitization is dependent on a TRAF2 binding site within thecytoplasmic domain of CD30 and cellular degradation of TRAF2 iscoincident with the onset of apoptosis.

2.3 Role of CD30 in the Immune System

2.3.1 Normal Immune System

The in vivo function of CD30 is not clearly understood. Ligation of CD30in vitro has been shown to induce either cellular proliferation orgrowth inhibition (Gruss et al., 1994, Blood. 83, 2045-2056). Ligationof CD30 on T cells has been demonstrated to regulate a variety of invitro T cell functions. Anti-CD30 mAbs co-stimulate with T cell antigenreceptor ligation to augment cellular proliferation in T cells that havebeen primed by either antigens (Del Prete et al., 1995, J. Exp. Med.,182:1655-1661) or anti-CD3 antibodies (Gilfillan et al., 1998, J.Immunol. 160:2180-2187). CD30 signaling also promotes cytokineproduction in T cells. Expression of interleukin (IL)-2. IL-4, IL-5,IFNγ, and TNFα are enhanced by CD30 signals in PHA activated peripheralT cells (Grüss and Hermann, 1996, Leukemia Lymphoma, 20:397-409), Thelper (Th) clones (Del Prete et al., 1995, J. Exp. Med., 182:1655-1661;Bengtsson et al., 2000, Scand. J. Immunol. 5′, 595-601), cytotoxic Tcell (Tc/CTL) lines (Bowen et al., 1996, J. Immunol. 156:442-449), andγδ T cells (Biswas et al., 2000, Eur. J. Immunol. 30:2172-2180). CD30may also play an important role in stimulating HIV replication.Cross-linking of CD30 by mAbs induces NF-κB activation and HIVproduction by a T cell line chronically infected with HIV (Biswas etal., 1995, Immunity, 2, 587-596). CD4⁺, HIV-infected T cells derivedfrom patients also respond to anti-CD30 mAbs or CD30L stimulation toproduce HIV (Maggi et al., 1995, Immunity 3:251-255).

CD30 has been implicated in the activation-induced cell death of Tcells. Ligation of a CD8-CD30 chimera expressed in a T cell hybridomaenhanced apoptosis mediated by the T cell antigen receptor (Lee et al.,1996, J. Exp. Med. 183:669-674). In a second model of agonist withdrawalinduced apoptosis in murine CD8 T cells, blockage of CD30 signalingusing a CD30-Ig fusion protein partially prevented CD8 T cells fromundergoing apoptosis (Telford et al., 1997, Cell. Immunol. 182:125-136). Results obtained from animal models also suggest a role ofCD30 in the induction of apoptosis in thymocytes, a negative selectionprocess for the deletion of auto-reactive T cells (Amakawa et al., 1996,Cell 84:551-562). In CD30^(−/−) null mice negative selection is severelydiminished, giving rise to increased thymocyte numbers (Amakawa et al.,1996 Cell 84:551-562); one demonstrated consequence being thatCD30-deficient CD8-positive T cells are orders of magnitude moreautoreactive in their capacitor to cause autoimmune diabetes (Kurts etal., 1999, Nature 398:341-344). Conversely, overexpression of CD30 hasbeen shown to promote programmed cell death in thymocytes, and henceenhances negative selection of auto-reactive T cells in the thymus(Chiarle et al., 1999, J. Immunol. 163:194-205), and prevent T cellautoresponses to non-lymphoid tissue in the periphery (Heath et al.,1999, Immunol. Rev. 169:23-29).

2.3.2 The Disease State

Because CD30 was demonstrated to be preferentially expressed on human Tcell clones secreting Th₂ cytokines (Del Prete et al., 1995, FASEB J.,9, 81-86), and cross-linking of CD30 on T cells promoted the developmentof Th₂-like T cells (Del Prete et al., 1995, J. Exp. Med., 182,1655-1661), it has been postulated that immunological disordersinvolving Th₂ cytokines may result from the dysregulation of CD30⁺ Tcells.

The expression of CD30 has been shown to be increased or altered in avariety of autoimmune and inflammatory diseases including atopic allergy(atopic dermatitis, atopic asthma, rhinoconjunctivitis, allergicrhinitis), systemic lupus erythematosus, systemic sclerosis(scleroderma), graft versus host disease (GVHD), HIV and EBV infection,measles, Omenn's syndrome, ulcerative colitis, rheumatoid arthritis,multiple sclerosis, psoriasis, Hashimoto's thyroiditis, primary biliarycirrhosis, Sjogren's syndrome, Wegener's granulomatosis, andtuberculosis (Gruss et al., 1997, Immunol. Today 18:156-163; Horie andWatababe, 1998, Sem. Immunol. 10:457-470; Bengtsson, 2001, Allergy56:593-603; Gerli et al., 2001, Trends Immunol. 22:72-77).

CD30+ infiltrating T cells have been found in lesions and inflamed sitesof atopic dermatitis (Caproni et al., 1997, Allergy 52:1063-1070;Cavagni et al., 2000, Allergy Immunol. 121:224-228), atopic asthma(Blanco Quirós et al., 1999, Pediatr. Allergy Immunol. 10:235-240), GVHD(D'Elios et al., 1997, J. Lewukoc. Biol. 61:539-544), tuberculosis (Munket al., 1997, Int. Immunol. 9:713-720), primary biliary cirrhosis(Harada et al., 1999, J. Gastroenterol. Hepatol. 14:1197-1202), measles(Vinante et al., 1999, Haematologica 84:683-689), rheumatoid arthritis(Gerli et al., 2000, J. Immunol. 164:4399-4407; Gerli et al., 1995,Clin. Exp. Immunol. 102:547-550), and psoriasis (Ferenczi et al., 2000,J. Autoimmun. 14:63-78). Although CD30 expression is strictly restrictedto a small percentage of activated lymphocytes in normal situations,increased expression of CD30 mRNA has been reported in atopic asthma andallergic rhinitis (Esnault et al., 1996, Clin. Exp. Immunol. 106:67-72).Correlation between altered expression of CD30 and immune disorders isfurther underscored by the high plasma levels of soluble CD30 (sCD30)found in many of the aforementioned diseases (Gruss et al., 1997.Immunol. Today 18:156-163; Horie and Watababe, 1998, Sem. Immunol.10:457-470; Bengtsson, 2001, Allergy 56:593-603; Gerli et al., 2001,Trends Immunol. 22:72-77). In some of these diseases high plasma levelsof sCD30 are not accompanied with circulating CD30⁺ T cells, butexpansion of CD30′ T cells in the disease tissue is believed to be apotential source of sCD30 (Mavilia, 1997, Am. J. Pathol. 151:1751-1758).

Several diseases have been demonstrated to involve both Th₂ cytokinesand CD30⁺ T cells (D'Elios et al., 1997, J. Leukoc. Biol. 61:539-544).Most patients with atopic dermatitis show increased production of Th₂cytokines with parallel increases in the serum IgE levels and augmentednumbers of circulating eosinophils (Leung, 1995, J. Allergy Clin.Immunol. 96:302-319). An increased frequency of allergen-specific Th₂cells producing IL-4, IL-5, and IL-13 can also be detected in theperipheral blood of atopic dermatitis patients (Kimura et al., 1998, J.Allergy Clin. Immunol., 101, 84-89). A correlation between CD30expression and Th₂ cytokine production has been noted in atopicdermatitis patients which has led to the suggestion that CD30 expressionin circulating T cells might serve as an in vivo marker for theTh₂-dominated condition (Yamamoto et al., 2000, Allergy, 55, 1011-1018).Allergen induced IL-4 production in atopic asthma correlates with CD30expression in PBMC isolated from atopic asthma patients (Leonard et al.,1997, Am. J. Respir. Cell Mol. Biol., 17, 368-375). This has also beenshown in bronchial alveolar lavage yδ T cells from asthma patients(Spinozzi et al., 1995, Mol. Med. 1:82.1-826). Likewise, in systemicsclerosis, a disease characterized by elevated serum levels of sCD30 andCD30 expression in skin lesions, IL-4 mRNA can be detected by in situhybridization in lymphocytes infiltrating skin lesions while most CD4⁺ Tcell clones generated from skin infiltrates also demonstrate a Th₂cytokine profile (Mavalia etal., 1997, Am. J. Path. 15, 1751-1758). Inpatients infected with HIV, a disproportionately higher frequency ofCD8⁺/CD30⁺ cells producing Th₂ cytokines could be detected in peripheralblood when compared to healthy donors (Manetti et al. 1994, J. Exp. Med.180:2407-2411). The emergence of a Th₂-type immunity in HIV infectedpatients also appears to be correlated with the immunopathologyassociated with disease progression (Rizzardi et al., 1998, Clin. Exp.Immunol. 114:61-65).

In contrast to the Th₂-related diseases, the role of CD30⁺ inTh₁-related diseases is not as well defined. High levels of sCD30 arefound in the CSF of patients with multiple sclerosis and the circulationof rheumatoid arthritis patients (McMillan et al., 2000, Acta Neurol.Scand. 101:239-243; Gerli et al., 2000, J. Immunol. 164:4399-4407). Inthese diseases, both Th₁ cytokine driven responses, high circulatinglevels of CD30 are associated with disease remission, suggesting thatTh₂ cytokines contributed by CD30⁺ T cells may counteract the pathogenicactivities of Th₁ cytokines (McMillan et al., 2000, Acta Neurol. Scand.101:239-243; Gerli et al., 2000, J. Immunol. 164:4399-4407). More recentexperiments examining CD30 expression on T cell subsets determined thatupon cellular activation Th₁ cells also express CD30 (Hamman et al.,1996, J. Immunol. 156: 1387-1391; Bengtsson et al., 1995, J. Leukoc.Biol. 58:683-689). An agonistic anti-CD30 mAb can stimulate theproduction of the Th₁ cytokine IFNγ in CD30⁺ Th₁ cells (Bengtsson etal., 2000, Scad. J. Immunol. 52:595-601). In atopic dermatitis, evidenceis available to demonstrate a shift from the predominance of Th₂cytokine production by T cell infiltrating skin lesions to theco-expression of Th₂ and Th₁ cytokines during disease progression (Greweet al., 1998, Immunol. Today 19:359). Hence CD30 T cells may also play arole in the production of Th₁ cytokines in Th₁-related diseases.

2.4 Immunotherapeutics

Sustained activation of lymphocytes against self-antigens or allergensand the failure to terminate ongoing immune responses subsequent to theclearance of antigens are the underlying causes of the pathologies seenin autoimmune diseases, allergic reactions, and chronic inflammatoryreactions. A variety of therapeutic regimens including antimetabolites,steroids, and anti-inflammatory agents are available for the treatmentof autoimmune, allergic, and inflammatory diseases. Although these drugsare efficacious in alleviating symptoms, none of them work byspecifically eliminating the pathogenic cells and most of them havesevere side effects on patients.

Elimination or attenuation of activated lymphocytes bearing CD30 couldbe efficacious in controlling autoimmune and chronic inflamatorydiseases. However, despite the above evidence for a role of CD30 inimmune disorders, targeting CD30 by mAb has not been demonstrated to beeffective in treating such disorders. Agents that are capable ofeliminating or attenuating CD30-bearing activated lymphocytes would behighly desirable in the treatment of immunological disorders.

3. SUMMARY OF THE INTENTION

The present invention is based on the surprising discovery of a novelactivity associated with certain classes of anti-CD30 antibodies. Thenovel activity is the ability of anti-CD30 antibodies to kill or inhibitthe growth of activated lymphocytes, in certain instances by signalingthrough the CD30 receptor pathway. In certain preferred embodiments, theantibodies of the invention are able to induce apoptosis or growtharrest of activated lymphocytes as monospecific antibodies, in theabsence of conjugation to cytotoxic reagents, and/or in the absence ofcells other than the CD30-expressing lymphocytes (e.g., in the absenceof effector cells).

Accordingly, the present invention provides methods for the treatment ofan immunological disorder in a subject, preferably wherein the immunedisorder is not cancer, comprising administering to the subject, in anamount effective for said treatment, a pharmaceutical compositioncomprising (a) a first antibody that (i) immunospecifically binds CD30and (ii) induces CD30 signaling in a lymphocyte and/or exerts acytostatic or cytotoxic effect on an activated lymphocyte; and (b) apharmaceutically acceptable carrier. The antibody can be human,humanized or chimeric. In one embodiment, the antibody is multivalent.In a preferred embodiment, the antibody competes for binding to CD30with monoclonal antibodies AC10 or HeFi-1.

The present invention further provides methods for the treatment of animmunological disorder in a subject, preferably wherein the immunedisorder is not cancer, comprising administering to the subject, in anamount effective for said treatment, a pharmaceutical compositioncomprising (a) a first antibody that (i) immunospecifically binds CD30and (ii) induces CD30 signaling in a lymphocyte and/or exerts acytostatic or cytotoxic effect on an activated lymphocyte; and (b) apharmaceutically acceptable carrier, said methods further comprisingadministering an agent that enhances or potentiates the cytostatic orcytotoxic effect of the first antibody. In certain embodiments, theagent that potentiates the cytostatic or cytotoxic effect of the firstantibody is a second antibody, a ligand that binds to a receptor orreceptor complex expressed on activated lymphocytes, or animmunosuppressive agent. Examples of such reagents and methods of theiruse are further described infra.

The present invention thus provides methods for the treatment of animmunological disorder in a subject, preferably wherein the immunedisorder is not cancer, comprising administering to the subject, in anamount effective for said treatment, a pharmaceutical compositioncomprising (a) a first antibody that (i) immunospecifically binds CD30and (ii) induces CD30 signaling in a lymphocyte and/or exerts acytostatic or cytotoxic effect on an activated lymphocyte; and (b) apharmaceutically acceptable carrier; said methods further comprisingadministering a second antibody to the subject. In a preferredembodiment, the second antibody recognizes a non-CD30 receptor orreceptor complex expressed on activated lymphocytes.

In certain specific embodiments, the present invention provides methodsfor the treatment of an immunological disorder in a subject, wherein theimmunological disorder is not cancer, comprising administering to thesubject, in an amount effective for said treatment, a pharmaceuticalcomposition comprising (a) an antibody that (i) immunospecifically bindsCD30 and (ii) competes for binding to CD30 with monoclonal antibody AC10or HeFi-1; and (b) a pharmaceutically acceptable carrier.

In other specific embodiments, the present invention provides methodsfor the treatment of an immunological disorder in a subject, wherein theimmunological disorder is not cancer, comprising administering to thesubject, in an amount effective for said treatment, a pharmaceuticalcomposition comprising (a) an antibody that (i) immunospecifically bindsCD30 and (ii) comprises SEQ ID NO:2 (the heavy chain variable region ofthe anti-CD30 antibody AC10); and (b) a pharmaceutically acceptablecarrier.

In other specific embodiments, the present invention provides methodsfor the treatment of an immunological disorder in a subject, wherein theimmunological disorder is not cancer, comprising administering to thesubject, in an amount effective for said treatment, a pharmaceuticalcomposition comprising (a) an antibody that (i) immunospecifically bindsCD30 and (ii) comprises one, two or all of: SEQ ID NO:4, SEQ ID NO:6 andSEQ ID NO:8 (the heavy chain CDRs of the anti-CD30 antibody AC10), orvariants of SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:8 that differ fromSEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:8 by one, two or three aminoacids; and (b) a pharmaceutically acceptable carrier.

In other specific embodiments, the present invention provides methodsfor the treatment of an immunological disorder in a subject, wherein theimmunological disorder is not cancer, comprising administering to thesubject, in an amount effective for said treatment, a pharmaceuticalcomposition comprising (a) an antibody that (i) immunospecifically bindsCD30 and (ii) comprises SEQ ID NO: 18 (the heavy chain variable regionof the anti-CD30 antibody HeFi-1); and (b) a pharmaceutically acceptablecarrier.

In other specific embodiments, the present invention provides methodsfor the treatment of an immunological disorder in a subject, wherein theimmunological disorder is not cancer, comprising administering to thesubject, in an amount effective for said treatment, a pharmaceuticalcomposition comprising (a) an antibody that (i) immunospecifically bindsCD30 and (ii) comprises one, two or all of: SEQ ID NO:20, SEQ ID NO:22and SEQ ID NO:24 (the heavy CDRs of the anti-CD30 antibody HeFi-1), orvariants of SEQ ID NO:20, SEQ ID NO:22 and SEQ ID NO:24 that differ fromSEQ ID NO:20, SEQ ID NO:22 and SEQ ID NO:24 by one, two or three aminoacids; and (b) a pharmaceutically acceptable carrier.

In yet other specific embodiments, the present invention providesmethods for the treatment of an immunological disorder in a subject,wherein the immunological disorder is not cancer, comprisingadministering to the subject, in an amount effective for said treatment,a pharmaceutical composition comprising (a) an antibody that (i)immunospecifically binds CD30 and (ii) competes for binding to CD30 withmonoclonal antibody AC10 or HeFi-1, wherein said antibody is conjugatedto a cytotoxic agent; and (b) a pharmaceutically acceptable carrier.

In certain specific embodiment of the present invention, the anti-CD30antibody is an agonistic antibody. In another specific embodiment of thepresent invention, the anti-CD30 antibody is not a non-agonisticantibody. In another specific embodiment, the anti-CD30 antibody doesnot block binding of CD30 ligand to CD30.

In a preferred embodiment, where a second antibody that recognizes anon-CD30 receptor or receptor complex is administered to the subject,such an antibody is capable of enhancing the cytotoxic or cytostaticeffect of the CD30 antibody. While not bound by any theory, such asecond antibody enhances the cytotoxic or cytostatic effect of the CD30antibody by delivering a cytostatic or cytotoxic signal to the activatedlymphocytes. Exemplary receptors or receptor complexes include animmunoglobulin gene superfamily member, a TNF receptor superfamilymember, an integrin, a cytokine receptor, a chemokine receptor, a majorhistocompatibility protein, a lectin, or a complement control protein.Non-limiting examples of suitable immunoglobulin superfamily members areCD2, CD3, CD4, CD8, CD19, CD22, CD28, CD79, CD90, CD152/CTLA-4. PD-1,and ICOS. Non-limiting examples of suitable TNF receptor superfamilymembers are CD27, CD40, CD95/Fas, CD134/OX40, CD137/4-1BB, TNF-R1,TNFR-2, RANK, TACI, BCMA, osteoprotegerin, Apo2/TRAIL-R1, TRAIL-R2,TRAIL-R3, TRAIL-R4, and APO-3. Non-limiting examples of suitableintegrins are CD11a, CD11b, CD11c, CD18, CD29, CD41, CD49a, CD49b,CD49c, CD49d, CD49e, CD49f, CD103, and CD104. Non-limiting examples ofsuitable lectins are C-type, S-type, and I-type lectin.

In certain preferred embodiments, antibodies useful in the presentmethods, i.e., antibodies that bind to CD30 and exert a cytostatic orcytotoxic effect on an activated lymphocyte, are bispecific antibodies.In a specific embodiment, the bispecific antibodies bind to both CD30and a non-CD30 receptor or receptor complex expressed on activatedlymphocytes. Preferably, the portion of the bispecific antibody thatbinds to the non-CD30 receptor or receptor complex is capable ofenhancing the cytotoxic or cytostatic effect of the CD30 antibody. Thenon-CD30 binding portion of the bispecific antibody preferably enhancesthe cytotoxic or cytostatic effect of the CD30 antibody by delivering acytostatic or cytotoxic signal to the activated lymphocytes. Thereceptor or receptor complex can comprise an immunoglobulin genesuperfamily member, a TNF receptor superfamily member, an integrin, acytokine receptor, a chemokine receptor, a major histocompatibilityprotein, a lectin, or a complement control protein. Non-limitingexamples of suitable immunoglobulin superfamily members are CD2, CD3,CD4, CD8, CD19, CD22, CD28, CD79, CD90, CD152/CTLA-4, PD-1, and ICOS.Non-limiting examples of suitable TNF receptor superfamily members areCD27, CD40, CD95/Fas, CD134/OX40, CD137/4-1BB, TNF-R1, TNFR-2, RANK,TACI, BCMA, osteoprotegerin. Apo2/TRAIL-R1, TRAIL-R1, TRAIL-R3,TRAIL-R4, and APO-3. Non-limiting examples of suitable integrins areCD11a, CD11b, CD11 c, CD18. CD29, CD41, CD49a, CD49b, CD49c, CD49d,CD49e, CD49f, CD103, and CD104. Non-limiting examples of suitablelectins are C-type, S-type, and I-type lectin.

In other embodiments, the therapeutic methods of the present inventionfurther comprise administering to the subject a ligand that binds to areceptor or receptor complex expressed on activated lymphocytes,concurrently or successively with the anti-CD30 antibody. Preferably,the ligand is capable of enhancing the cytotoxic or cytostatic effect ofthe CD30 antibody, for example by delivering a cytostatic or cytotoxicsignal to the activated lymphocytes. The receptor or receptor complexcan comprise an immunoglobulin gene superfamily member, a TNF receptorsuperfamily member, an integrin, a cytokine receptor, a chemokinereceptor, a major histocompatibility protein, a lectin, or a complementcontrol protein. Non-limiting examples of suitable immunoglobulinsuperfamily members are CD2, CD3, CD4, CD8, CD19, CD22, CD28, CD79,CD90, CD152/CTLA-4, PD-1, and ICOS. Non-limiting examples of suitableTNF receptor superfamily members are CD27, CD40, CD95/Fas, CD134/OX40,CD137/4-1BB, TNF-R1, TNFR-2, RANK, TACI. BCMA, osteoprotegerin,Apo2/TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4, and APO-3. Non-limitingexamples of suitable integrins are CD11a, CD11b, CD11c, CD18, CD29,CD41, CD49a, CD49b, CD49c, CD49d CD49e, CD49f, CD103, and CD104.Non-limiting examples of suitable lectins are C-type, S-type, and I-typelectin. Ligands that bind to the foregoing receptors are known to thoseof skill in the art.

In certain specific embodiments of the present therapeutic methods, theanti-CD30 antibody is a fusion protein comprising the amino acidsequence of a second protein that is not an antibody. In a preferredembodiment, the second protein confers multivalent binding properties tothe CD30 antibody.

In other embodiments, the therapeutic methods of the present inventionfurther comprise administering to the subject a cytostatic, cytotoxic,and/or immunosuppressive agent. In one embodiment, the immunosuppressiveagent is gancyclovir, acyclovir, etanercept, rapamycin, cyclosporine ortacrolimus. In other embodiments, the immunosuppressive agent is anantimetabolite, a purine antagonist (e.g. azathioprine or mycophenolatemofetil), a dihydrofolate reductase inhibitor (e.g., methotrexate), aglucocorticoid. (e.g., cortisol or aldosterone), or a glucocorticoidanalogue (e.g., prednisone or dexamethasone). In yet other embodiments,the immunosuppressive agent is an alkylating agent (e.g.,cyclophosphamide). In yet other embodiments, the immunosuppressive agentis an anti-inflamatory agent, including but not limited to acyclooxygenase inhibitor, a 5-lipoxygenase inhibitor, and a leukotrienereceptor antagonist.

In certain preferred embodiments, antibodies useful in the presentmethods, i.e., antibodies that bind to CD30 and induce CD30 signaling ina lymphocyte and/or exert a cytostatic or cytotoxic effect on anactivated lymphocyte, are conjugated to a cytostatic, cytotoxic orimmunosuppressive agent. Such conjugated antibodies are sometimesreferred to herein as anti-CD30 antibody-drug conjugates (“ADC” or“ADCs”) or anti-CD30 antibody-cytotoxic agent/immunosuppressive agentconjugates.

In certain preferred embodiments, the cytotoxic agent is selected fromthe group consisting of an enediyne, a lexitropsin, a duocarmycin, ataxane, a puromycin, a dolastatin, a maytansinoid, and a vinca alkaloid.In certain, more specific embodiments, the cytotoxic agent ispaclitaxel, docetaxel, CC-1065, SN-38, topotecan,morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin,dolastatin-10, echinomycin, combretastatin, calicheamicin, maytansine,DM-1, auristatin E, AEB, AEVB, AEFP, MMAE, or netropsin. The structuresof AEB, AEVB, AEFP and MMAE are depicted in Section 3.1, infra.

In other preferred embodiments, the cytotoxic agent of an anti-CD30antibody-cytotoxic agent conjugate of the invention is an anti-tubulinagent. In more specific embodiments, the cytotoxic agent is selectedfrom the group consisting of a vinca alkaloid, a podophyllotoxin, ataxane, a baccatin derivative, a cryptophysin, a maytansinoid, acombretastatin, and a dolastatin. In more specific embodiments, thecytotoxic agent is vincristine, vinblastine, vindesine, vinorelbine,VP-16, camptothecin, paclitaxel, docetaxel, epithilone A, epithilone B,nocodazole, coichicine, colcimid, estramustine, cemadotin,discodermolide, maytansine, DM-1, AEFP, auristatin E, AEB, AEVB, AEFP,MMAE or eleutherobin.

In a specific embodiment, the cytotoxic agent of an anti-CD3 Gantibody-cytotoxic agent conjugate of the invention is MMAE. In anotherspecific embodiment, the cytotoxic agent of an anti-CD30antibody-cytotoxic agent conjugate of the invention is AEFP.

In specific embodiments, the anti-CD30 antibody of an anti-CD30antibody-cytotoxic agent conjugate of the invention is conjugated to thecytotoxic agent via a linker, wherein the linker is peptide linker. Inspecific embodiments, the anti-CD30 antibody of an anti-CD30antibody-cytotoxic agent conjugate of the invention is conjugated to thecytotoxic agent via a linker, wherein the linker is a val-cit linker, aphe-lys linker, a hydrazone linker, or a disulfide linker. In certainembodiments, the anti-CD30 antibody of an anti-CD30 antibody-cytotoxicagent conjugate of the invention is conjugated to the cytotoxic agentvia a peptide linker.

In certain embodiments, the conjugate of the invention isanti-CD30-valine-citrulline-MMAE (anti-CD30-val-citMMAE oranti-CD30-vcMMAE) or anti-CD30-valine-citrulline-AEFP(anti-CD30-val-citAEFP or anti-CD30-vcAEFP). In specific embodiments,the conjugate of the invention is AC10-valine-citrulline-MMAE(AC10-val-citMMAE or AC10-vcMMAE) or AC10-valine-citrulline-AEFP(AC10-val-citAEFP or AC10-vcAEFP).

In other embodiments, the conjugate of the invention isanti-CD30-phenylalanine-lysine-MMAE (anti-CD30-phe-lysMMAE oranti-CD30-fkMMAE) or anti-CD30-phenylalanine-lysine-AEFP(anti-CD30-phe-lysAEFP or anti-CD30-fkAEFP). In specific embodiments,the conjugate of the invention is AC10-phenylalanine-lysine-MMAE(AC10-phe-lysMMAE or AC10-fkMMAE) or AC10-phenylalanine-lysine-AEFP(AC10-phe-lysAEFP or AC10-fkAEFP).

Thus, in a specific embodiment, the present invention provides methodsfor the treatment of an immunological disorder in a subject, wherein theimmunological disorder is not cancer, comprising administering to thesubject, in an amount effective for said treatment, a pharmaceuticalcomposition comprising (a) cAC10-val-cit-MMAE; and (b) apharmaceutically acceptable carrier.

In another specific embodiment, the present invention provides methodsfor the treatment of an immunological disorder in a subject, wherein theimmunological disorder is not cancer, comprising administering to thesubject, in an amount effective for said treatment, a pharmaceuticalcomposition comprising (a) cAC10-val-cit-AEFP; and (b) apharmaceutically acceptable carrier.

In certain embodiments, the anti-CD30 antibody of an anti-CD30antibody-cytotoxic agent conjugate of the invention is conjugated to thecytotoxic agent via a linker, wherein the linker is hydrolyzable at a pHof less than 5.5. In a specific embodiment the linker is hydrolyzable ata pH of less than 5.0.

In certain embodiments, the anti-CD30 antibody of an anti-CD30antibody-cytotoxic agent conjugate of the invention is conjugated to thecytotoxic agent via a linker, wherein the linker is cleavable by aprotease. In a specific embodiment, the protease is a lysosomalprotease. In other specific embodiments, the protease is, inter alia, amembrane-associated protease, an intracellular protease, or an endosomalprotease.

In certain embodiments, the anti-CD30 antibody of the invention is amonoclonal antibody, a humanized chimeric antibody, a chimeric antibody,a humanized antibody, a glycosylated antibody, a multispecific antibody,a human antibody, a single-chain antibody, a Fab fragment, a F(ab′)fragment, a F(ab′)₂ fragment, a Fd, a single-chain Fv, adisulfide-linked Fv, a fragment comprising a V_(L) domain, or a fragmentcomprising a V_(H) domain. In certain embodiments, the anti-CD30antibody of an anti-CD30 antibody-cytotoxic agent conjugate of theinvention is a polypeptide that binds specifically to CD30. In certainembodiments, the antibody is a bispecific antibody. In otherembodiments, the antibody is not a bispecific antibody.

In certain embodiments, the anti-CD30 antibody is radioactively labeled.In certain embodiments, the anti-CD30 antibody of the anti-CD30antibody-cytotoxic agent conjugate is radioactively labeled. In specificembodiments, the radioactive label is ⁹⁰Y, ¹¹¹In, ²¹¹At, ¹³¹I, ²¹²Bi,²¹³Bi, ²²⁵Ac, ¹⁸⁶Re, ¹⁸⁵Re, ¹⁰⁹Pd, ⁶⁷Cu, ⁷⁷Br, ¹⁰⁵Rh, ¹⁹⁸Au, ¹⁹⁹Au or²¹²Pb.

Immunological disorders encompassed by the methods of the presentinvention are Th₂-lymphocyte related disorders (e.g., atopic dermatitis,systemic lupus erythematosus (“SLE”), atopic asthma,rhinoconjunctivitis, allergic rhinitis, Omenn's syndrome, systemicsclerosis, and chronic graft versus host disease); Th₁lymphocyte-related disorders (e.g., rheumatoid arthritis, multiplesclerosis, psoriasis, Sjorgren's syndrome, Hashimoto's thyroiditis,Grave's disease, primary biliary cirrhosis, Wegener's granulomatosis,tuberculosis and acute graft versus host disease); viralinfection-related disorders (e.g., Epstein-Barr virus, humanimmunodeficiency virus, human T leukemia virus, hepatitis B virus, andmeasles virus infections); and activated B lymphocyte-related disorders(e.g., systemic lupus erythematosus, Goodpasture's syndrome, rheumatoidarthritis, and tripe I diabetes).

3.1 Abbreviations

The abbreviation “AEFP” refers todimethylvaline-valine-dolaisoleuine-dolaproine-phenylalanine-p-phenylenediamine,the auristatin E derivative depicted below:

The abbreviation “MMAE” refers to monomethyl auristatin E, theauristatin E derivative depicted below:

The abbreviation “AEB” refers to an ester produced by reactingauristatin E with paraacetyl benzoic acid, the structure of which isdepicted below:

The abbreviation “AEVB” refers to an ester produced by reactingauristatin E with benzoylvaleric acid, the structure of which isdepicted below:

The abbreviations “fk” and “phe-lys” refer to the linkerphenylalanine-lysine.

The abbreviations “vc” and “val-cit” refer to the linkervaline-citrulline.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Expression of CD30 on Jurkat T cells: The Jurkat T cell line wasexamined for the expression of CD3, CD4, CD28, and CD30 by flowcytometric analysis.

FIG. 2. Effect of anti-CD30 on the proliferation of Jurkat T cell:Jurkat T cells were incubated with graded doses of a chimeric AC10(cAC10) anti-CD30 mAb with (XL cAC10) or without (cAC10) the presence ofa secondary cross-linking goat anti-human (GAH) Fcγ specific Ab.Proliferation was assessed by a pulse of tritiated thymidine (³H-TdR)during the last 4 hours of a 72 hour incubation.

FIG. 3. Secondary cross-linking of anti-CD30 mAb on the Jurkat T cellsinhibited proliferation: Jurkat T cells were incubated with graded dosesof the AC10 or HeFi-1 mAbs in the presence of a secondary cross-linkinggoat anti-mouse (GAM) Fcγ specific Ab at different primary to secondaryAb ratios as indicated in the figure. Proliferation was assessed by apulse of ³H-TdR during the last 4 hours of a 48-hour incubation.

FIG. 4. Secondary cross-linking of anti-CD30 mAb on the Jurkat T cellsinduced apoptosis: Jurkat T cells were treated with 0.2 μg/ml of eitherAC10 or HeFi-1 cross-linked by 0.8 μg/ml of a GEM secondary Ab. Cellcycle disposition and DNA sysnthesis were detected by propidium iodide(PI) and anti-bromodeoxyuridine (BrdU) staining after 24 and 48 hours oftreatment.

FIG. 5. Secondary cross-linking of anti-CD30 mAb on the Jurkat T cellsinduced apoptosis: Jurkat T cells were treated with graded doses ofeither AC10 or HeFi-1 cross-linked by a GAM secondary Ab at a primary tosecondary Ab concentration ratio of 1:4. Cell cycle disposition and DNAsynthesis were detected by PI and anti-BrdU staining after 24 and 48hours of treatment.

FIG. 6. Anti-CD3 mAb enhanced anti-CD30-induced apoptosis in Jurkat Tcells: Jurkat T cells were treated with AC10, the anti-CD3 mAb OKT3, ora combination of both mAbs at graded doses as indicated in the figure.Four-fold excess of a GAM secondary Ab was used to cross-link theprimary mAbs. Cell cycle disposition and DNA sysnthesis were detected byPI and anti-BrdU staining after 24 and 48 hours of treatment.

FIG. 7. Detection of anti-CD30-induced apoptosis in Jurkat T cells byAnnexin V binding: Jurkat T cells were treated with AC10, HeFi-1, theanti-CD3 mAb OKT3, or a combination of AC10 or HeFi-1 and OKT3.Anti-CD30 and anti-CD3 mAbs were used at 2 μg/ml. GAM secondary Ab wasused in 10-fold excess to cross-link the primary mAbs. Binding ofFITC-conjugated Annexin V enabled the detection of cells undergoingapoptosis. Membrane permeability to PI was used to detect dead cellsthat had lost membrane integrity. In the figure, Annexin V⁻/PI⁻ eventsrepresent live cells (lower left quadrant), Annexin V⁺/PI⁻ eventsrepresent apoptotic cells (lower right quadrant), and Annexin V⁺/PI⁺events represent dead cells (upper right quadrant). Numbers outside ofthe density plots denote the percentage of cells present in each of thequadrants.

FIG. 8. Chemical structures of the antibody drug conjugates (ADCs)cAC10-vcMMAE, cAC10-fkMMAE, cAC10-vcAEFP, and cAC10-fkAEFP.

FIG. 9. Growth inhibitory effect of the cAC10-vcMMAE conjugate on theproliferation of Jurkat T cells: Graded doses of the cAC10-vcMMAEconjugate or a non-binding control IgG (cIgG)-vcMMAE conjugate wereadded to Jurkat T cells at the initiation of culture. Cells were exposedto the ADCs continuously for a total of 96 hr. Proliferation wasassessed by a pulse of H-TdR during the last 16 hours of incubation.

FIG. 10. CD30 induction on activated normal peripheral blood mononuclearcells (PBMC): PBMC from normal donors were stimulated with eitheranti-CD3, anti-CD3+ anti-CD28, or in medium alone (negative control). Ondays 0, 2, 4, 6, and 8 cells were harvested and expression of CD30 wasdetermined on both CD4 cells (shown) and CD8 cells (not shown) bymulti-color flow cytometric analysis.

FIG. 11. Growth inhibitory effects of cAC10 ADCs on activated normalhuman PBMC: PBMC from normal donors were activated withanti-CD3+anti-CD8 mAbs as described in FIG. 10. Graded doses ofdifferent ADCs as indicated in the figure were added to the cells at theinitiation of culture. Cells were exposed to the ADCs continuously for atotal of 48 or 72 hr. Proliferation was assessed by incorporation of³H-TdR during the last 16 hours of incubation.

FIG. 12. Induction of CD30 on memory and naïve T cells: Peripheral bloodT lymphocytes, memory T lymphocytes, and naïve T lymphocytes wereenriched from PBMC using immuno-selection. T cells were then activatedby anti-CD3+anti-CD28 mAbs for 72 hr in the presence of both recombinanthuman interleukin (rhIL)-2 and rhIL-4. Expression of CD30 on CD4⁺ andCD8⁺ cells was then assessed by flow cytometry.

FIG. 13. Growth inhibitory effects of cAC10 ADCs on activated memory andnaïve T lymphocytes: Memory and naïve T cells enriched from PBMC wereinduced to express CD30 as described in FIG. 12. After 72 hr ofinduction, T cells were harvested and treated with graded doses ofdifferent ADCs as indicated in the figure in the presence of rhIL-2.Cells were exposed to the ADCs continuously for an additional 48 or 72hr. Proliferation was assessed by incorporation of ³H-TdR during thelast 16 hours of incubation.

FIG. 14. CD30 induction on T cells stimulated by allogeneic cells:CD4-enriched PBMC were stimulated with successive cycles of irradiatedallogeneic Burkitt's lymphoma Daudi cells. Expression of CD30 on CD4cells was determined by flow cytometric analysis 3-5 days and 7-9 daysafter the addition of the allogeneic stimulator cells.

FIG. 15. Generation of T lymphocyte clones: a schematic to summarize thegeneration of T cell clones from PBMC.

FIG. 16. Phenotype of T lymphocyte clones: Ten independent T lymphocyteclones isolated from 3 different normal donors according to the schemedepicted in FIG. 15 were examined for their surface expression of CD3,CD4, CD8, CD28, and CD30 by flow cytometric analysis. Histograms fromtwo representative clones are shown, and the levels of receptorexpression in all 10 clones indicated by the mean fluorescenceintensities obtained from flow cytometric analysis were tabulated.

FIG. 17. Cytokine expression by T lymphocyte clones: T lymphocyte clonesdepicted in FIG. 16 were examined for their ability of express IL-2, 4,5, 13, IFNγ, and TNFα by flow cytometric analysis. Histograms from tworepresentative clones are shown, and the levels of cytokine expressionin all 10 clones indicated by the mean fluorescence intensities obtainedfrom flow cytometric analysis were tabulated. T lymphocytes clones werealso assigned to different subsets based on cytokine expression in thebottom row of the table

FIG. 18. Upregulation of CD30 upon stimulation of T lymphocyte clones: Aresting Th₂ clone and a resting Tc₂ clone representing the panel shownin FIGS. 16 and 17 were stimulated with phytohemaggutinin (PHA),irradiated feeder cells, IL-2, and IL-4. CD25 and CD30 expression ondays 0, 2, 4, and 7 were determined by flow cytometric analysis.

FIG. 19. Growth inhibitory effects of cAC10 ADCs on the T lymphocteclone 40E10: 40E10 cells were induced to express CD30 as described inFIG. 18. At the peak of CD30 expression on day 2 of activation, cellswere harvested and treated with graded doses of different ADCs asindicated in the figure in the presence of rhIL-2 and rhIL-4. Cells wereexposed to the ADCs continuously for an additional 48 or 72 hr.Proliferation was assessed by incorporation of ³H-TdR during the last 16hours of incubation.

FIG. 20. Growth inhibitory effects of cAC10 ADCs on the T lymphocyteclone 40H7: 40H7 cells were induced to express CD30 as described in FIG.1S. At the peak of CD30 expression on day 2 of activation, cells wereharvested and treated with graded doses of different ADCs as indicatedin the figure in the presence of rhIL-2 and rhIL-4. Cells were exposedto the ADCs continuously for an additional 48 or 72 hr. Proliferationwas assessed by a pulse of ³H-TdR during the last 16 hours ofincubation.

FIG. 21. Inhibition of proliferation in T cell clones induced by cAC10ADCs was accompanied by apoptosis induction: Clone 40E10 and 40H7 wereincubated with 1 μg/ml of different ADCs as indicated in the figure.After 48 hr of incubation, Annexin V binding and permeability of PI wereused to assess the extent of apoptosis induction.

FIG. 22. Growth inhibitory effect of cAC10 ADCs on the T lymphocyteclones 3.27.2 and 4.01.1: 3.27.2 and 4.01.1 cells were induced toexpress CD30 as described in FIG. 18. At the peak of CD30 expression onday 2 of activation, cells were harvested and treated with graded dosesof different ADCs as indicated in the figure in the presence of rhIL-2.Cells were exposed to the ADCs continuously for an additional 72 hr.Proliferation was assessed by a pulse of ³H-TdR during the last 4 hoursof incubation.

FIG. 23. A summary of the efficacies of cAC10 ADCs to inhibit theproliferation of CD30⁺ T lymphocyte clones and activated normal Tlymphocytes.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the use of proteins that bind to CD30and induce CD30 signaling in a lymphocyte and/or exert a cytostatic orcytotoxic effect on activated lymphocytes to treat or preventimmunological disorders. The invention further relates to proteins thatcompete with AC10 or HeFi-1 for binding to CD30 and induce CD30signaling in a lymphocyte and/or exert a cytostatic or cytotoxic effecton activated lymphocytes. In one embodiment, the protein is an antibody.In a preferred mode of the embodiment, the antibody is AC10 or HeFi-1,most preferably a humanized or chimeric form of AC10 or HeFi-1.

In certain embodiments, the anti-CD30 antibodies of the presentinvention are capable of inducing apoptosis or growth arrest ofactivated lymphocytes as monospecific antibodies, in the absence ofconjugation to cytotoxic reagents (e.g. small molecules, toxins,radioactive isotopes), and/or in the absence of cells other than theCD30-expressing lymphocytes (e.g., in the absence of effector cells suchas natural killer cells). Without being bound by any theory, the presentinventors believe that such antibodies induce apoptosis or growth arrestby signaling through the CD30 pathway.

The invention further relates to the use of proteins encoded by andnucleotide sequences of AC10 and HeFi-1 genes to treat or preventimmunological disorders. The invention further relates to fragments andother derivatives and analogs of such AC10 and HeFi-1 proteins. Nucleicacids encoding such fragments or derivatives are also within the scopeof the invention. Production of the foregoing proteins, e.g., byrecombinant methods, is provided.

The invention also relates to the use of AC10 and HeFi-1 proteins andderivatives including fusion/chimeric proteins which are functionallyactive, i.e., which are capable of displaying binding to CD30 andexerting a cytostatic or cytotoxic effect on activated lymphocytes, totreat or prevent immunological disorders.

Antibodies to CD30 whose use is encompassed by the invention includehuman, chimeric or humanized antibodies, and such antibodies conjugatedto cytotoxic agents such as chemotherapeutic drugs.

The invention further relates to methods of treating or preventingimmunological disorders comprising administering a compositioncomprising a protein or nucleic acid of the invention alone or incombination with a cytotoxic agent, including but not limited to achemotherapeutic drug.

For clarity of disclosure, and not by way of limitation, the detaileddescription of the invention is divided into the subsections whichfollow.

5.1 Proteins of the Invention

The present invention encompasses the use of proteins, including but notlimited to antibodies, that bind to CD30 and exert cytostatic and/orcytotoxic effects on activated lymphocytes, for the treatment of animmunological disorder. The invention further relates to the use ofproteins that compete with AC10 or HeFi-1 for binding to CD30 and exerta cytostatic or cytotoxic effect on activated lymphocytes for thetreatment of immunological disorders.

The present invention further encompasses the use of proteins comprisinga CDR of HeFi-1 (SEQ ID NO:20, SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:28,SEQ ID NO:30 or SEQ ID NO:32) or AC10 (SEQ ID NO:4; SEQ ID NO:6; SEQ IDNO:8; SEQ ID NO:12; SEQ ID NO:14; or SEQ ID NO:16) for the treatment ofan immunological disorder.

The present invention further encompasses the use of proteins comprisinga variable region of HeFi-1 (SEQ ID NO: 18 or SEQ ID NO:26) or AC10 (SEQID NO:2 or SEQ ID NO: 10) for treating an immunological disorder. Atable indicating the region of AC10 or HeFi-1 to which each SEQ ID NOcorresponds to is provided below: TABLE 1 NUCLEOTIDE OR MOLECULE AMINOACID SEQ ID NO AC10 Heavy Chain Variable Region Nucleotide 1 AC10 HeavyChain Variable Region Amino Acid 2 AC10 Heavy Chain-CDR1(H1) Nucleotide3 AC 10 Heavy Chain-CDR1(H1) Amino Acid 4 AC 10 Heavy Chain-CDR2(H2)Nucleotide 5 AC 10 Heavy Chain-CDR2(H2) Amino Acid 6 AC 10 HeavyChain-CDR3(H3) Nucleotide 7 AC 10 Heavy Chain-CDR3(H3) Amino Acid 8 AC10 Light Chain Variable Region Nucleotide 9 AC 10 Light Chain VariableRegion Amino Acid 10 AC 10 Light Chain-CDR1(L1) Nucleotide 11 AC 10Light Chain-CDR1(L1) Amino Acid 12 AC 10 Light Chain-CDR2(L2) Nucleotide13 AC 10 Light Chain-CDR2(L2) Amino Acid 14 AC 10 Light Chain-CDR3(L3)Nucleotide 15 AC 10 Light Chain-CDR3(L3) Amino Acid 16 HeFi-1 HeavyChain Variable Region Nucleotide 17 HeFi-1 Heavy Chain Variable RegionAmino Acid 18 HeFi-1 Heavy Chain-CDR1(H1) Nucleotide 19 HeFi-1 HeavyChain-CDR1(H1) Amino Acid 20 HeFi-1 Heavy Chain-CDR2(H2) Nucleotide 21HeFi-1 Heavy Chain-CDR2(H2) Amino Acid 22 HeFi-1 Heavy Chain-CDR3(H3)Nucleotide 23 HeFi-1 Heavy Chain-CDR3(H3) Amino Acid 24 HeFi-1 LightChain Variable Region Nucleotide 25 HeFi-1 Light Chain Variable RegionAmino Acid 26 HeFi-1 Light Chain-CDR1(L1) Nucleotide 27 HeFi-1 LightChain-CDR1(L1) Amino Acid 28 HeFi-1 Light Chain-CDR2(L2) Nucleotide 29HeFi-1 Light Chain-CDR2(L2) Amino Acid 30 HeFi-1 Light Chain-CDR3(L3)Nucleotide 31 HeFi-1 Light Chain-CDR3(L3) Amino Acid 32

The present invention further comprises the use of functionalderivatives or analogs of AC10 and HeFi-1 for treating immunologicaldisorders. As used herein, the term “functional” in the context of apeptide or protein of the invention indicates that the peptide orprotein is 1) capable of binding to CD30 and 2) induces CD30 signalingin a lymphocyte and/or exerts a cytostatic and/or cytotoxic effect onactivated lymphocytes.

Generally, antibodies suitable for practicing the methods of the presentinvention immunospecifically bind CD30 and induce CD30 signaling in alymphocyte and/or exert cytostatic and cytotoxic effects on activatedlymphocytes. Antibodies suitable for practicing the methods of theinvention are preferably monoclonal, and may be multispecific, human,humanized or chimeric antibodies, single chain antibodies, Fabfragments, F(ab′) fragments, fragments produced by a Fab expressionlibrary, and CD30 binding fragments of any of the above. The term“antibody,” as used herein, refers to immunoglobulin molecules andimmunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site that immunospecificallybinds CD30. The immunoglobulin molecules of the invention can be of anytype (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2,IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.

In certain embodiments of the invention, the antibodies are humanantigen-binding antibody fragments of the present invention and include,but are not limited to, Fab, Fab′ and F(ab′)₂, Fd, single-chain Fvs(scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) andfragments comprising either a V_(L) or V_(H) domain. Antigen-bindingantibody fragments, including single-chain antibodies, may comprise thevariable region(s) alone or in combination with the entirety or aportion of the following: hinge region, CH1, CH2, CH3 and CL domains.Also included in the invention are antigen-binding fragments alsocomprising any combination of variable region(s) with a hinge region,CH1, CH2, CH3 and CL domains. Preferably, the antibodies are human,murine (e.g., mouse and rat), donkey, sheep, rabbit, goal, guinea pig,camelid, horse, or chicken. As used herein, ‘human ’ antibodies includeantibodies having the amino acid sequence of a human immunoglobulin andinclude antibodies isolated from human immunoglobulin libraries, fromhuman B cells, or from animals transgenic for one or more humanimmunoglobulin, as described infra and, for example in U.S. Pat. No.5,939,598 by Kucherlapati et al.

The antibodies suitable for practicing the methods of the presentinvention may be monospecific, bispecific, trispecific or of greatermultispecificity. Multispecific antibodies may be specific for differentepitopes of CD30 or may be specific for both CD30 as well as for aheterologous protein. See, e.g., PCT publications WO 93/17715; WO92/08802; WO 91/00360; WO 92/05793; Tutt, et al., 1991, J. Immunol.147:60-69; U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920;5,601,819; Kostelny et al., 1992, J. Immunol. 148:1547-1553.Multispecific antibodies, including bispecific and trispecificantibodies, useful for practicing the present invention are antibodiesthat immunospecifically bind to both CD30 (including but not limited toantibodies that have the CDRs and/or heavy chains of the monoclonalantibodies Ki-2, Ki-4, Ki-5, Ki-7, Ber-H2, HRS-1, HRS-4, Ki-1, Ki-6,M67, Ki-3, M44, HeFi-1, and AC10) and a lymphocyte surface receptor orreceptor complex, such as an immunoglobulin gene superfamily member, aTNF receptor superfamily member, an integrin, a cytokine receptor, achemokine receptor, a major histocompatibility protein, a lectin(C-type, S-type, or I-type), or a complement control protein. Examplesof such molecules, and antibodies against such molecules from which abispecific antibody can be derived, are provided in Section 5.12.2,infra. In a preferred embodiment, the binding of the portion of themultispecific antibody to the lymphocyte cell surface molecule ormolecular complex enhances the cytotoxic or cytostatic effect of theanti-CD30 antibody by delivering a cytostatic or cytotoxic signal to theactivated lymphocytes.

In certain specific embodiment of the present invention, the anti-CD30antibody is an agonistic antibody. In another specific embodiment of thepresent invention, the anti-CD30 antibody is not a non-agonisticantibody. In another specific embodiment, the anti-CD30 antibody doesnot block binding of CD30 ligand to CD30.

Antibodies useful in the present methods may be described or specifiedin terms of the particular CDRs they comprise. In certain embodimentsantibodies of the invention comprise one or more CDRs of AC10 and/orHeFi-1. The invention encompasses the use of an antibody or derivativethereof comprising a heavy or light chain variable domain, said variabledomain comprising (a) a set of three CDRs, in which said set of CDRs arefrom monoclonal antibody AC10 or HeFi-1, and (b) a set of four frameworkregions, in which said set of framework regions differs from the set offramework regions in monoclonal antibody AC10 or HeFi-1, respectively,and in which said antibody or derivative thereof immunospecificallybinds CD30 and induces CD30 signaling in a lymphocyte and/or exerts acytotoxic or cytostatic effect on activated lymphocytes.

In a specific embodiment, the invention encompasses the use of anantibody or derivative thereof for treating an immunological disorder,wherein the antibody comprises a heavy chain variable domain, saidvariable domain comprising (a) a set of three CDRs, in which said set ofCDRs comprises SEQ ID NO:4, 6, or 8 and (b) a set of four frameworkregions, in which said set of framework regions differs from the set offramework regions in monoclonal antibody AC10, and in which saidantibody or derivative thereof immunospecifically binds CD30, andinduces CD30 signaling in a lymphocyte and/or exerts a cytotoxic orcytostatic effect on activated lymphocytes.

In a specific embodiment, the invention encompasses the use of anantibody or derivative thereof for the treatment of an immunologicaldisorder, wherein the antibody comprises a heavy chain variable domain,said variable domain comprising (a) a set of three CDRs, in which saidset of CDRs comprises SEQ ID NO:20, 22 or 24 and (b) a set of fourframework regions, in which said set of framework regions differs fromthe set of framework regions in monoclonal antibody HeFi-1, and in whichsaid antibody or derivative thereof immunospecifically binds CD30, andinduces CD30 signaling in a lymphocyte and/or exerts a cytotoxic orcytostatic effect on activated lymphocytes.

In a specific embodiment, the invention encompasses the use of anantibody or derivative thereof for the treatment of an immunologicaldisorder, wherein the antibody comprises a light chain variable domain,said variable domain comprising (a) a set of three CDRs, in which saidset of CDRs comprises SEQ ID NO: 12, 14 or 16, and (b) a set of fourframework regions, in which said set of framework regions differs fromthe set of framework regions in monoclonal antibody AC10, and in whichsaid antibody or derivative thereof immunospecifically binds CD30, andinduces CD30 signaling, in a lymphocyte and/or exerts a cytotoxic orcytostatic effect on activated lymphocytes.

In a specific embodiment, the invention encompasses using an antibody orderivative thereof for the treatment of an immunological disease,wherein the antibody comprises a light chain variable domain, saidvariable domain comprising (a) a set of three CDRs, in which said set ofCDRs comprises SEQ ID NO:28, 30, or 32, and (b) a set of four frameworkregions, in which said set of framework regions differs from the set offramework regions in monoclonal antibody HeFi-1, and in which saidantibody or derivative thereof immunospecifically binds CD30, andinduces CD30 signaling in a lymphocyte and/or exerts a cytotoxic orcytostatic effect on activated lymphocytes.

Additionally, antibodies that may be used in the methods of the presentinvention may also be described or specified in terms of their primarystructures. Antibodies having at least 50%, at least 55%, at least 60%,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95% and most preferably at least 98% identity (ascalculated using methods known in the art and described herein) to thevariable regions and AC10 or HeFi-1 are also included in the presentmethods for treating immunological disorders. Antibodies useful in themethods of the present invention may also be described or specified interms of their binding affinity to CD30. Preferred binding affinitiesinclude those with a dissociation constant or Kd less than 5×10⁻² M,10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M,10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M,10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×⁻¹³ M, 10⁻¹³ M,5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M.

The antibodies of the invention, i.e., antibodies that are useful fortreating immunological disorders, include derivatives that are modified,i.e., by the covalent attachment of any type of molecule to the antibodysuch that covalent attachment does not prevent the antibody from bindingto CD30 or from exerting a cytostatic or cytotoxic effect on activatedlymphocytes. For example, but not by way of limitation, the antibodyderivatives include antibodies that have been modified, e.g., byglycosylation, acetylation, pegylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolytic),ticcleavage, linkage to a cellular ligand or other protein, etc. Any ofnumerous chemical modifications may be carried out by known techniques,including, but not limited to specific chemical cleavage, acetylation,formylation, metabolic synthesis of turicamycin, etc. Additionally, thederivative may contain one or more non-classical amino acids.

The antibodies that may be used in the treatment of immunologicaldisorders may be generated by any suitable method known in the art.Polyclonal antibodies to CD30 can be produced by various procedures wellknown in the art. For example, CD30 can be administered to various hostanimals including, but not limited to, rabbits, mice, rats, etc. toinduce the production of sera containing polyclonal antibodies specificfor the protein. Various adjuvants may be used to increase theimmunological response, depending on the host species, and include butare not limited to, Freund's (complete and incomplete), mineral gelssuch as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and corynebacteriumparvum. Such adjuvants are also well known in the art.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.,1988); Hammerling, et al., in: Monoclonal Antibodies and T-CellHybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporatedby reference in their entireties). The term “monoclonal antibody” asused herein is not limited to antibodies produced through hybridomatechnology. The term “monoclonal antibody” refers to an antibody that isderived from a single clone, including any eukaryotic, prokaryotic, orphage clone, and not the method by which it is produced.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and resell known in the art. In anon-limiting example, mice can be immunized with CD30 or a cellexpressing CD30 or a fragment or derivative thereof. Once an immuneresponse is detected, e.g. antibodies specific for CD3′ are detected inthe mouse serum, the mouse spleen is harvested and splenocytes isolated.The splenocytes are then fused by well known techniques to any suitablemyeloma cells, for example cells from cell line SP20 available from theATCC. Hybridomas are selected and cloned by limited dilution. Thehybridoma clones are then assayed by methods known in the art for cellsthat secrete antibodies capable of binding CD30 and exerting a cytotoxicor cytostatic effect on activated lymphocytes. Ascites fluid, whichgenerally contains high levels of antibodies, can be generated byinjecting mice with positive hybridoma clones.

Antibody fragments which recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)₂ fragments of theinvention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)₂ fragments). F(ab′)₂ fragments contain thevariable region, the light chain constant region and the CH 1 domain ofthe heavy chain.

For example, antibodies useful in the methods of the present inventioncan also be generated using various phage display methods known in theart. In phage display methods, functional antibody domains are displayedon the surface of phage particles which carry the nucleic acid sequencesencoding them. In a particular embodiment, such phage can be utilized todisplay antigen binding domains expressed from a repertoire orcombinatorial antibody library (e.g. human or murine). In phage displaymethods, functional antibody domains are displayed on the surface ofphage particles which carry the nucleic acid sequences encoding them. Inparticular, DNA sequences encoding V_(H) and V_(L) domains are amplifiedfrom animal cDNA libraries (e.g., human or murine cDNA libraries oflymphoid tissues). The DNA encoding the V_(H) and V_(L) domains arerecombined together with an scFv linker by PCR and cloned into aphagemid vector (e.g., p CANTAB 6 or pComb 3 HSS). The vector iselectroporated in E. coli and the E. coli is infected with helper phage.Phage used in these methods are typically filamentous phage including fdand M13 binding domains expressed from phage with Fab, Fv or disulfidestabilized Fv antibody domains recombinantly fused to either the phagegene III or gene VIII protein. Phage expressing an antigen bindingdomain that binds to CD30 or an AC10 or HeFi-binding portion thereof canbe selected or identified with antigen e.g., using labeled antigen orantigen bound or captured to a solid surface or bead. Examples of phagedisplay methods that can be used to make the antibodies of the presentinvention include those disclosed in Brinkman et al., 1995, J. Immunol.Methods 182:41-50; Ames et al., 1995, J. Immunol. Methods 184:177-186;Kettleborough et al., 1994, Eur. J. Immunol. 24:952-958; Persic et al.,1997, Gene 187:9-18; Burton et al., 1994, Advances in Immunology,191-280; PCT Application No. PCT/GB91/O1 134; PCT Publications WO90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/1 1236; WO95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409;5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108;each of which is incorporated herein by reference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)₂ fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., BioTechniques 1992,12(6):864-869; and Sawai et al., 1995, AJRI 34:26-34; and Better et al.,1988, Science 240:1041-1043 (said references incorporated by referencein their entireties).

Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al., 1991, Methods in Enzymology 203:46-88; Shu etal., 1993, PNAS 90:7995-7999; and Skerra et al., 1988, Science240:1038-1040. For some uses, including in vivo use of antibodies inhumans and in vitro proliferation or cytotoxicity assays, it ispreferable to use chimeric, humanized, or human antibodies. A chimericantibody is a molecule in which different portions of the antibody arederived from different animal species, such as antibodies having avariable region derived from a murine monoclonal antibody and a humanimmunoglobulin constant region. Methods for producing chimericantibodies are known in the art. See e.g., Morrison, Science, 1985,229:1202; Oi et al., 1986, BioTechniques 4:214; Gillies et al., 1989, J.Immunol. Methods 125:191-202; U.S. Pat. Nos. 5,807,715; 4,816,567; and4,816,397, which are incorporated herein by reference in their entirety.Humanized antibodies are antibody, molecules from non-human speciesantibodies that bind the desired antigen having one or more CDRs fromthe non-human species and framework and constant regions from a humanimmunoglobulin molecule. Often, framework residues in the humanframework regions will be substituted with the corresponding residuefrom the CDR donor antibody to alter, preferably improve, antigenbinding. These framework substitutions are identified by methods wellknown in the art, e.g., by modeling of the interactions of the CDR andframework residues to identify framework residues important for antigenbinding and sequence comparison to identify unusual framework residuesat particular positions. (See, e.g., Queen et al., U.S. Pat. No.5,585,089; Riechmann et al., 1988, Nature 332:323, which areincorporated herein by reference in their entireties.) Antibodies can behumanized using a variety of techniques known in the art including, forexample, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S.Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing(EP 592,106; EP 519,596; Padlan, Molecular Immunology, 1991,28(4/5):489-498; Studnicka et al., 1994, Protein Engineering7(6):805-814; Roguska. et al., 1994, PNAS 91:969-973), and chainshuffling (U.S. Pat. No. 5,565,332).

Completely human antibodies are particularly desirable for thetherapeutic treatment of human patients. Human antibodies can be made bya variety of methods known in the art including phage display methodsdescribed above using antibody libraries derived from humanimmunoglobulin sequences. See also, U.S. Pat. Nos. 4,444,887 and4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893,WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which isincorporated herein by reference in its entirety.

Human antibodies can also be produced using transgenic mice whichexpress human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells. The mouseheavy and light chain immunoglobulin genes may be renderednon-functional separately or simultaneously with the introduction ofhuman immunoglobulin loci by homologous recombination. In particular,homozygous deletion of the JH region prevents endogenous antibodyproduction. The modified embryonic stem cells are expanded andmicroinjected into blastocysts to produce chimeric mice. The chimericmice are then bred to produce homozygous offspring which express humanantibodies. The transgenic mice are immunized in the normal fashion witha selected antigen, e.g., all or a portion of CD30. Monoclonalantibodies directed against the antigen can be obtained from theimmunized, transgenic mice using conventional hybridoma technology. Thehuman immunoglobulin transgenes harbored by the transgenic micerearrange during B cell differentiation, and subsequently undergo classswitching and somatic mutation. Thus, using such a technique, it ispossible to produce therapeutically useful IgG, IgA, IgM and IgEantibodies. For an overview of this technology for producing humanantibodies, see, Lonberg and Huszar, 1995, Int. Rev. Immunol. 13:65-93.For a detailed discussion of this technology for producing humanantibodies and human monoclonal antibodies and protocols for producingsuch antibodies, see, e.g., PCT publications WO 98/24893; WO 92/01047;WO 96/34096; WO 96/33735; European Patent No. 0 598 877; U.S. Pat. Nos.5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806;5,814,318; 5,885,793; 5,916,771; and 5,939,598, which are incorporatedby reference herein in their entirety. In addition, companies such asAbgenix, Inc. (Freemont, Calif.) and Medarex (Princeton, N.J.) can beengaged to provide human antibodies directed against a selected antigenusing technology similar to that described above.

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., 1994, Bio/technology12:899-903).

Further, antibodies to CD30 can, in turn, be utilized to generateanti-idiotype antibodies that “mimic” proteins of the invention usingtechniques well known to those skilled in the art. (See, e.g. Greenspan& Bona, 1989, FASEB J. 7(5):437-444; and Nissinoff. 1991, J. Immunol.147(8):2429-243S). Fab fragments of such anti-idiotypes can be used intherapeutic regimens to elicit an individual's own immune responseagainst CD30 present on activated lymphocytes.

As alluded to above, proteins that are therapeutically orprophylactically useful against activated lymphocytes need not beantibodies. Accordingly, proteins of the invention may comprise one ormore CDRs from an antibody that binds to CD30 and induces CD30 signalingin a lymphocyte and/or exerts a cytotoxic and or cytostatic effect onactivated lymphocytes. Preferably, a protein of the invention is amultimer, most preferably a dimer. As used herein, a “protein of theinvention” is a protein, including but not limited to an antibody, thatbinds to CD30, and induces CD30 signaling in a lymphocyte and/or exertsa cytotoxic or cytostatic effect on activated lymphocytes.

The invention also provides methods of treating immunological disordersusing proteins, including but not limited to antibodies, thatcompetitively inhibit binding of AC10 or HeFi-1 to CD30 as determined byany method known in the art for determining competitive binding, forexample, the immunoassays described herein. In preferred embodiments,the protein competitively inhibits binding of AC10 or HeFi-1 to CD30 byat least 50%, more preferably at least 60%, yet more preferably at least70%, and most preferably at least 75%. In other embodiments, the proteincompetitively inhibits binding of AC10 or HeFi-1 to CD30 by at least80%, at least 85%, at least 90%, or at least 95%.

As discussed in more detail below, the present invention providesmethods of treating immunological disorders using proteins, includingantibodies, that bind to activated lymphocytes and exert a cytostatic orcytotoxic effect on the lymphocytes. The proteins can be administeredeither alone or in combination with other compositions in the preventionor treatment of immunological disorders. The proteins may further berecombinantly fused to a heterologous protein at the N- or C-terminus orchemically conjugated (including covalently and non-covalentlyconjugations) to cytotoxic agents, proteins or other compositions. Forexample, antibodies of the present invention may be recombinantly fusedor conjugated to molecules useful as chemotherapeutics or toxins, orcomprise a radionuclide for use as a radio-therapeutic. See, e.g., PCTpublications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No.5,314,995; and EP 396,387.

Proteins useful in the present methods may be produced recombinantly bfusing the coding region of one or more of the CDRs of an antibody ofthe invention in frame with a sequence coding for a heterologousprotein. The heterologous protein may provide one or more of thefollowing characteristics: added therapeutic benefits; promote stableexpression; provide a means of facilitating high yield recombinantexpression; or provide a multimerization domain.

In addition to proteins comprising one or more CDRs of an antibody thatbinds to CD30, and induces CD30 signaling in a lymphocyte and/or exertsa cytotoxic or cytostatic effect on lymphocytes, proteins that areuseful in the therapeutic methods of the invention may be identifiedusing any method suitable for screening for protein-proteininteractions. Initially, proteins are identified that bind to CD30, thentheir ability to exert a cytostatic or cytotoxic effect on activatedlymphocytes can be determined. Among the traditional methods which canbe employed are “interaction cloning” techniques which entail probingexpression libraries with labeled CD30 in a manner similar to thetechnique of antibody probing of λgt11 libraries, supra) a. By way ofexample and not limitation, this can be achieved as follows: a cDNAclone encoding CD30 (or an AC10 or HeFi-1 binding domain thereof) ismodified at the terminus by inserting the phosphorylation site for theheart muscle kinase (HMK) (Blanar & Rutter, 1992, Science256:1014-1018). The recombinant protein is expressed in E. coli andpurified on a GDP-affinity column to homogeneity (Edery et al., 1988,Gene 74:517-525) and labeled using γ³²P-ATP and bovine heart musclekinase (Sigma) to a specific activity of 1×10⁸ cpm/μg, and used toscreen a human placenta λgt11 cDNA library in a “far-Western assay”(Blanar & Rutter, 1992, Science 256:1014-1018). Plaques which interactwith the CD30 probe are isolated. The cDNA inserts of positive λ plaquesare released and subcloned into a vector suitable for sequencing, suchas pBluescript KS (Stratagene).

One method which detects protein interactions is in vivo, the two-hybridsystem, is described in detail for illustration purposes only and not byway of limitation. One version of this system has been described (Chienet al., 1991, Proc. Natl. Acad. Sci. USA, 88:9578-9582) and iscommercially available from Clontech (Palo Alto, Calif.).

Once a CD30-binding protein is identified, its ability (alone or whenmultimerized or fused to a dimerization or multimerization domain) toelicit a cytostatic or cytotoxic effect on activated lymphocytes isdetermined by the methods described in Section 5.7, infra.

Without limitation as to mechanism of action, a protein that binds toCD30 and induces CD30 signaling in a lymphocyte and/or exerts acytostatic or cytotoxic effect on activated lymphocytes preferably hasmore than one CD30-binding site and therefore a capacity to cross linkCD30 molecules on the surface of an activity ated lymphocyte. Proteinswhich bind to CD30 or compete for binding to CD30 with AC10 or HeFi-1can acquire the ability, to induce cytostatic or cytotoxic effects onactivated lymphocytes if dimerized or multimerized. Where theCD30-binding protein is a monomeric protein, it can be expressed intandem, thereby resulting in a protein with multiple CD30 binding sites.The CD30-binding sites can be separated by a flexible linker region. Inanother embodiment, the CD30-binding proteins can be chemicallycross-linked, for example using gluteraldehyde, prior to administration.In a preferred embodiment, the CD30-binding region is fused with aheterologous protein, wherein the heterologous protein comprises adimerization and multimerization domain. Prior to administration of theprotein of the invention to a subject for the purpose of treating orpreventing immunological disorders, such a protein is subjected toconditions that allows formation of a homodimer or heterodimer. Aheterodimer, as used herein, may comprise identical dimerization domainsbut different CD30-binding regions, identical CD30-binding regions butdifferent dimerization domains, or different CD30-binding regions anddimerization domains.

Particularly preferred dimerization domains are those that originatefrom transcription factors.

In one embodiment, the dimerization domain is that of a basic regionleucine zipper (“bZIP”). bZIP proteins characteristically possess twodomains—a leucine zipper structural domain and a basic domain that isrich in basic amino acids, separated by a “fork” domain (C. Vinson etal., 1989, Science, 246:911-916). Two bZIP proteins dimerize by forminga coiled coil region in which the leucine zipper domains dimerize.Accordingly, these coiled coil regions may be used as fusion partnersfor proteins that will be useful in the therapeutic methods describedherein.

Particularly useful leucine zipper domain are those of the yeasttranscription factor GCN4, the mammalian transcription factorCCAAT/enhancer-binding protein C/EBP, and the nuclear transform inoncogene products, Fos and Jun (see Landschultz et al., 1988, Science240:1759-1764; Baxevanis and Vinson, 1993, Curr. Op. Gen. Devel.,3:278-285; and O'Shea et al., 1989, Science, 243:538-542).

In another embodiment, the dimerization domain is that of a basic-regionhelix-loop-helix (“bHLH”) protein (Murre et al., 1989, Cell,56:777-783). bHLH proteins are also composed of discrete domains, thestructure of which allows them to recognize and interact with specificsequences of DNA. The helix-loop-helix region promotes dimerizationthrough its amphipathic helices in a fashion analogous to that of theleucine zipper region of the bZIP proteins (Davis et al., 1990 Cell,60:733-746; Voronova and Baltimore, 1990 Proc. Natl. Acad. Sci. USA,87:4722-4726). Particularly useful hHLH proteins are myc, max, and mac.

Heterodimers are known to form between Fos and Jun (Bohmann et al.,1987, Science, 238:1386-1392), among members of the ATF/CREB family (Haiet al., 1989, Genes Dev., 3:2083-2090), among members of the C/EBPfamily (Cao et al., 1991, Genes Dev., 5:1538-1552; Williams et al, 1991,Genes Dev., 5:1553-1567; and Roman et al., 1990, Genes Dev.,4:1404-1415), and between members of the ATF/CREB and Fos/Jun familiesHai and Curran, 1991, Proc. Natl. Acad. Sci. USA, 88:3720-3724).Therefore, when a protein of the invention is administered to a subjectas a heterodimer comprising different dimerization domains, anycombination of the foregoing may be used.

In a preferred aspect, a proteins of the invention, including but notlimited to an antibody-drug conjugate of the invention, is substantiallypurified (e.g., substantially free from substances that limit its effector produce undesired side-effects). In certain specific embodiments, theprotein of the invention is 40% pure, more preferably about 50% pure,and most preferably about 60% pure. In certain specific embodiments, theprotein of the invention is approximately 60-65%, 65-70%, 70-75%,75-80%, 80-85%, 85-90%, 90-95%, or 95-98% pure. In another specificembodiment, the protein of the invention is approximately 99% pure.

5.1.1 Isolation of AC10 or HeFi-1 Genes

The invention relates to the use of AC10 or HeFi-1 nucleic acids, e.g.,for gene therapy of immunological disorders or for recombinantexpression of an antibody molecule that can be use for the treatment ofan immunological disorder. Accordingly, the invention provides purifiednucleic acids consisting of at least 8 nucleotides (i.e., a hybridizableportion) of an AC10 or HeFi-1 gene sequence in other embodiments, thenucleic acids consist of at least 25 (contiguous) nucleotides, 50nucleotides, 100, or 200 nucleotides of an AC10 or HeFi-1 sequence, or afull-length AC10 or HeFi-1 variable region coding sequence. In the sameor other embodiments, the nucleic acids are smaller than 50, 75, 100, or200 or 5000 nucleotides in length. Nucleic acids can be single or doublestranded. The invention also relates to nucleic acids hybridizable to orcomplementary to the foregoing sequences or their reverse complements,and in particular, such nucleic acids that encode proteins that bind toCD30, compete with AC10 or HeFi-1 for binding to CD30, and/or increasethe binding of CD30 ligand to CD30 by at least 45%, 50%, 60%, or 65%. Inspecific aspects, nucleic acids are provided which comprise a sequencecomplementary to at least 10, 25, 50, 100, or 200 nucleotides or theentire coding region of an AC10 or HeFi-1 variable region gene.

Nucleic acids encoding derivatives and analogs of AC10 or HeFi-1proteins are additionally provided.

5.1.2 Cloning Procedures

Specific embodiments for the cloning of an AC10 or HeFi-1 nucleic acidfollow. In a specific embodiment, total RNA is isolated from a mAb AC10or HeFi-1-producing hybridoma and polymerase chain reaction is used toamplify desired variable region sequences, using primers based on thesequences disclosed herein. By way of another example, mRNA is isolatedfrom a mAb AC10 or HeFi-1-producing hybridoma, cDNA is made and ligatedinto an expression vector (e.g., a bacteriophage derivative) such thatit is capable of being expressed by the host cell into which it is thenintroduced. Various screening assays can then be used to select for theexpressed product. In one embodiment, selection is on the basis ofhybridization to a labeled probe representing a portion of an AC10 orHeFi-1 gene or its RNA or a fragment thereof (Benton and Davis, 1977,Science 196:180; Grunstein and Hogness, 1975, Proc. Natl. Acad. Sci.U.S.A. 72:3961). Those DNA fragments with substantial homology to theprobe will hybridize. It is also possible to identify the appropriatefragment by restriction enzyme digestion(s) and comparison of fragmentsizes with those expected according to a known restriction map if suchis available. Further selection can be carried out on the basis of theproperties of the gene.

Alternatively, the presence of the desired gene may be detected byassays based on the physical, chemical, or immunological properties ofits expressed product. For example, cDNA clones, or DNA clones whichhybrid-select the proper mRNAs, can be selected and expressed to producea protein that has, e.g., similar or identical electrophoreticmigration, isoelectric focusing behavior, proteolytic digestion maps, orfunctional activity, as known for an AC10 or HeFi-1 protein. Forexample, ability to bind CD30 can be detected in an ELISA (enzyme-linkedimmunosorbent assay)-type procedure.

An AC10 or HeFi-1 gene can also be identified by mRNA selection usingnucleic acid hybridization followed by in vitro translation. In thisprocedure, fragments are used to isolate complementary mRNAs byhybridization. Functional assays (e.g., binding to CD30, etc.) of the invitro translation products of the isolated products of the isolatedmRNAs identifies the mRNA and, therefore, the complementary DNAfragments that contain the desired sequences.

In another embodiment, an AC10 or HeFi-1 nucleic, most preferably anAC10 or HeFi-1 nucleic acid encoding the heavy or light chain variablesregion or a heavy or light chain CDR, can be chemically synthesized fromthe sequences disclosed herein. For example, the AC10 and HeFi-1sequences can be synthesized by standard methods known in the art, e.g.,by use of an automated DNA synthesizer (such as those commerciallyavailable from Biosearch, Applied Biosystems, etc.). Alternatively, theAC10 or HeFi-1 nucleic acid may be synthesized by a commerciallyavailable service, for example by Blue Heron Biotechnology (Bothell,Wash.) or QIAGEN Inc. (Valencia, Calif.). Other methods of isolatingAC10 or HeFi-1 genes known to the skilled artisan can be employed.

The isolated AC10 or HeFi-1 nucleic acid (e.g. a nucleic acid encodingAC10 or HeFi-1 or one or more CDRs or variable regions thereof) can thenbe inserted into an appropriate cloning vector. A large number ofvector-host systems known in the art may be used. Possible vectorsinclude, but are not limited to, plasmids or modified viruses, but thevector system must be compatible with the host cell used. Such vectorsinclude, but are not limited to, bacteriophages such as lambdaderivatives, or plasmids such as PBR322 or pUC plasmid derivatives orthe Bluescript vector (Stratagene). The insertion into a cloning vectorcan, for example, be accomplished by ligating the DNA fragment into acloning vector which has complementary cohesive termini. However, if thecomplementary restriction sites used to fragment the DNA are not presentin the cloning vector, the ends of the DNA molecules may beenzymatically modified. Alternatively, any site desired may be producedby ligating nucleotide sequences (linkers) onto the DNA termini; theseligated linkers may comprise specific chemically synthesizedoligonucleotides encoding restriction endonuclease recognitionsequences. In an alternative method, the cleaved vector and an AC10 orHeFi-1 gene may be modified by homopolymeric tailing, or by PCR withprimers containing the appropriate sequences. Recombinant molecules canbe introduced into host cells via transformation, transfection,infection, electroporation, etc., so that many copies of the genesequence are generated. Antibodies comprising one or more CDRs from AC10or HeFi-1 and framework regions from a different immunoglobulin moleculecan be produced using a variety of techniques known in the artincluding, for example, CDR-grafting (EP 239,400; PCT publication WO91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneeringor resurfacing (EP 592,106; EP 519,596; Padlan, 1991, MolecularImmunology 28(4/5):489-498; Studnicka et al., 1.994, Protein Engineering7(6):805-814; Roguska et al., 1994, PNAS 91:969-973), and chainshuffling (U.S. Pat. No. 5,565,332).

In a preferred embodiment of the present invention, an AC10 or HeFi-1nucleic acid, for example a nucleic acid encoding an AC10 or HeFi-1heavy or light chain variable region, can be cloned into animmunoglobulin expression vector. Briefly, the AC10 or HeFi-1 nucleicacid is synthesized or otherwise obtained by any of the methodsdescribed herein, preferably flanked by appropriate restriction sites,then cloned into a vector suitable for expression of immunoglobulinmolecules. A number of vectors have been described that contain, forexample, sequences encoding immunoglobulin constant regions andrestriction sites suitable for in frame cloning of antibody variableregions operably linked to a promoter and optionally a signal sequenceuseful for expression in a desired host cell. Non-limiting examples ofvectors that have been designed for this purpose are described in McLeanet al., 2000, Mol Immunol. 37(14):837-45; Liang et al., 2001, J. ImmunolMethods 247(1-2):119-30; Persic et al., 1997, Gene 187(1):9-18; Skerra.1994 Gene 141(1):79-84; Walls et al., 1993, Nucleic Acids Res21(12):2921-29; Coloma et al., 1992, J. Immunol. Methods 152(1):89-104.These vectors or similarly designed vectors can be used for cloning AC10and HeFi-1 sequences for expression purposes.

In an alternative method, the desired gene may be identified andisolated after insertion into a suitable cloning vector in a “shot gun”approach. Enrichment for the desired gene, for example, by sizefractionization, can be done before insertion into the cloning vector.

In specific embodiments, transformation of host cells with recombinantDNA molecules that incorporate an isolated AC10 or HeFi-1 gene, cDNA, orsynthesized DNA sequence enables generation of multiple copies of thegene. Thus, the gene may be obtained in large quantities by growingtransformants, isolating the recombinant DNA molecules from thetransformants and, when necessary, retrieving the inserted gene from theisolated recombinant DNA.

The AC10 or HeFi-1 sequences provided by the instant invention includethose nucleotide sequences encoding substantially the same amino acidsequences as found in native AC10 or HeFi-1 variable regions, and thoseencoded amino acid sequences with functionally equivalent amino acids,as well as those encoding other AC10 or HeFi-1 derivatives or analogs,as described below for AC10 or HeFi-1 derivatives and analogs.

5.2 Binding Assays

As described above, the proteins, including antibodies, that are usefulfor the treatment of immunological disorders according to the methods ofthe present-invention bind to CD30 and exert a cytostatic or cytotoxiceffect on activated lymphocytes. Methods of demonstrating the ability,of a protein to bind to CD30 are described herein.

Antibodies may be assayed for immunospecific binding to CD30 by anymethod known in the art. The immunoassays which can be used include butare not limited to competitive and non-competitive assay systems usingtechniques such as Western blots, radioimmunoassays, ELISA (enzymelinked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few. Such assays areroutine and well known in the art (see, e.g., Ausubel et. al., eds.,1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons,Inc., New York, which is incorporated by reference herein in itsentirety). Exemplary immunoassays are described briefly below (but arenot intended by way of limitation).

Immunoprecipitation protocols generally comprise lysing a population ofcells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100,1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphateat pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/orprotease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate),adding the antibody to the cell lysate, incubating for a period of time(e.g., 1-4 hours) at 4° C., adding protein A and/or protein G sepharosebeads to the cell lysate, incubating for about an hour or more at 4° C.,washing the beads in lysis buffer and resuspending the beads inSDS/sample buffer. The ability of the antibody to immunoprecipitate CD30can be assessed by, e.g., Western blot analysis. One of skill in the artwould be knowledgeable as to the parameters that can be modified toincrease the binding of the antibody to CD30 and decrease the background(e.g., pre-clearing the cell lysate with sepharose beads). For furtherdiscussion regarding immunoprecipitation protocols see, e.g., Ausubel etal., eds., 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York at 10.16.1.

Western blot analysis generally comprises preparing protein samples,electrophoresis of the protein samples in a polyacrlamide eel (e.g.,8%-20% SDS-PAGE depending on the molecular weight of the antigen),transferring the protein sample from the polyacrylamide gel to amembrane such as nitrocellulose. PVDF or nylon, incubating the membranein blocking solution (e.g., PBS with 3% BSA or non-fat milk), washingthe membrane in washing buffer (e.g., PBS-Tween 20), blotting themembrane with primary antibody (i.e., the putative anti-CD30 antibody)diluted in blocking buffer, washing the membrane in washing buffer,incubating the membrane with a secondary antibody (which recognizes theprimary antibody, e.g., an anti-human antibody) conjugated to an enzymesubstrate (e.g., horseradish peroxidase or alkaline phosphatase) orradioactive molecule (e.g., ³²P or ¹²⁵I) diluted in blocking buffer,washing the membrane in wash buffer, and detecting the presence of thesecondary antibody. One of skill in the art would be knowledgeable as tothe parameters that can be modified to increase the signal detected andto reduce the background noise. For further discussion regarding Westernblot protocols see, e.g., Ausubel et al., eds., 1994, Current Protocolsin Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at10.8.1.

ELISAs comprise preparing antigen (i.e., CD30), coating the well of a 96well microtiter plate with the CD30, adding the antibody conjugated to adetectable compound such as an enzyme (e.g., horseradish peroxidase oralkaline phosphatase) to the well and incubating for a period of time,and detecting the presence of the antibody. In ELISAs the antibody doesnot have to be conjugated to a detectable compound; instead, a secondantibody (which recognizes the antibody of interest) conjugated to adetectable compound may be added to the well. Further, instead ofcoating the well with the antigen, the antibody may be coated to thewell. In this case, a second antibody conjugated to a detectablecompound may be added following the addition of CD30 protein to thecoated well. One of skill in the art would be knowledgeable as to theparameters that can be modified to increase the signal detected as wellas other variations of ELISAs known in the art. For further discussionregarding ELISAs see, e.g., Ausubel et al. eds., 1994, Current Protocolsin Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at11.2.1.

The binding affinity of an antibody to CD30 and the off-rate of anantibody CD30 interaction can be determined by competitive bindingassays. One example of a competitive binding assay is a radioimmunoassaycomprising the incubation of labeled CD30 (e.g., ³H or ¹²⁵I) with theantibody of interest in the presence of increasing amounts of unlabeledCD30, and the detection of the antibody bound to the labeled CD30. Theaffinity of the antibody for CD30 and the binding off-rates can then bedetermined from the data by Scatchard plot analysis. Competition with asecond antibody (such as AC10 or HeFi-1) can also be determined usingradioimmunoassays. In this case, CD30 is incubated with the antibody ofinterest conjugated to a labeled compound (e.g. ³H or ¹²⁵I) in thepresence of increasing amounts of an unlabeled second antibody.Alternatively, the binding affinity of an antibody to CD30 and the on-and off-rates of an antibody-CD30 interaction can be determined bysurface plasmon resonance.

Proteins that are useful in the methods of the invention may also beassayed for their ability to bind to CD30 by a standard assay known inthe art. Such assays include far Westerns and the yeast two hybridsystem. These assays are described in Section 5.2, supra. Anothervariation on the far Western technique described above entails measuringthe ability of a labeled candidate protein to bind to CD30 in a Westernblot. In one non-limiting example of a far Western blot, CD30 or thefragment thereof of interest is expressed as a fusion protein furthercomprising glutathione-S-transferase (GST) and a proteinserine/threonine kinase recognition site (such as a cAMP-dependentkinase recognition site). The fusion protein is purified onglutathione-Sepharose beads (Pharmacia Biotech) and labeled with bovineheart kinase (Sigma) and 100 μCi of ³²P-ATP (Amersham). The testprotein(s) of interest are separated by SDS-PAGE and blotted to anitrocellulose membrane, then incubated with the labeled CD30.Thereafter, the membrane is washed and the radioactivity quantitated.Conversely, the protein of interest can be labeled by the same methodand used to probe a nitrocellulose membrane onto which CD30 has beenblotted.

5.3 Sequences Related to AC10 and HeFi-1

The present invention further encompasses the use of proteins andnucleic acids comprising a region of homology to CDRs of AC10 andHeFi-1, or the coding regions therefor, respectively, for the treatmentor prevention of an immunological disorder. In various embodiments, theregion of homology is characterized by at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95% or at least 98% identity with thecorresponding region of AC10 or HeFi-1.

In one embodiment, the present invention provides a method of treatingor preventing an immunological disorder comprising administering to apatient in need thereof a protein with a region of homology to a CDR ofHeFi-1 (SEQ ID NO:20, SEQ ID NO:22′; SEQ ID NO:24; SEQ ID NO:28, SEQ IDNO:30 or SEQ ID NO:32), provided that the protein induces CD30 signalingin a lymphocyte and/or exerts a cytotoxic or cytostatic effect onactivated lymphocytes. In another embodiment, the present inventionprovides a method of treating or preventing an immunological disordercomprising administering to a patient in need thereof a protein with aregion of homology to a CDR of AC10 (SEQ ID NO:4; SEQ ID NO:6; SEQ IDNO:8; SEQ ID NO:12; SEQ ID NO:14; or SEQ ID NO:16), provided that theprotein induces CD30 signaling in a lymphocyte 90 and/or exerts acytotoxic or cytostatic effect on activated lymphocytes.

In another embodiment, the present invention provides a method oftreating or preventing an immunological disorder comprisingadministering to a patient in need thereof a nucleic acid with a regionof homology to a CDR coding region of HeFi-1 (SEQ ID NO:19, SEQ IDNO:21, SEQ ID NO:23, SEQ ID NO:27, SEQ ID NO:29 or SEQ ID NO:31),provided that the encoded protein induces CD30 signaling in a lymphocyteand/or exerts a cytotoxic or cytostatic effect on activated lymphocytes.In yet another embodiment, the present invention provides a method oftreating or preventing an immunological disorder comprisingadministering to a patient in need thereof a nucleic acid with a regionof homology to a CDR coding region of AC10 (SEQ ID NO:3, SEQ ID NO:5.SEQ ID NO:7, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15), provided thatthe encoded protein induces CD30 signaling in a lymphocyte and/or exertsa cytotoxic or cytostatic effect on activated lymphocytes.

The present invention further encompasses methods of treating orpreventing an immunological disorder comprising administering to apatient in need thereof a protein or nucleic acids comprising a regionof homology to the variable regions of AC10 and HeFi-1, or the codingregion therefor, respectively. In various embodiments, the region ofhomology is characterized by at least 50%, at least 55%, at least 60%,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95% or at least 98% identity with the correspondingregion of AC10 or HeFi-1.

In one embodiment, the present invention provides methods of treating orpreventing an immunological disorder comprising administering to apatient in need thereof a protein with a region of homology to avariable region of HeFi-1 (SEQ ID NO: 18 or SEQ ID NO: 26). In anotherembodiment, the present invention provides methods of treating orpreventing an immunological disorder comprising administering to apatient in need thereof a protein with a region of homology to avariable region of AC10 (SEQ ID NO: 2 or SEQ ID NO: 10).

In one embodiment, the present invention provides methods of treating orpreventing an immunological disorder comprising administering to apatient in need thereof a nucleic acid with a region of homology to avariable region coding region of HeFi-1 (SEQ ID NO:17 or SEQ ID NO:25).In another embodiment, the present invention provides methods oftreating or preventing an immunological disorder comprisingadministering to a patient in need thereof a nucleic with a region ofhomology to a variable region coding region of AC10 (SEQ ID NO:1 or SEQID NO:9).

To determine the percent identity of two amino acid sequences or of twonucleic acids, e.g. between the sequences of an AC10 or HeFi-1 variableregion and sequences from other proteins with regions of homology to theAC10 or HeFi-1 variable region, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in the sequence of afirst amino acid or nucleic acid sequence for optimal alignment with asecond amino or nucleic acid sequence). The amino acid residues ornucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % identity=# of identical positions/total # ofpositions (e.g., overlapping positions)×100). In one embodiment, the twosequences are the same length.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. A preferred, non-limitingexample of a mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl.Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul, 1993,Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm isincorporated into the NBLAST and XBLAST programs of Altschul, et al,1990, J. Mol. Biol. 215:403-410. BLAST nucleotide searches can beperformed with the NBLAST program, score=100, wordlength=12 to obtainnucleotide sequences homologous to a nucleic acid encoding a SCA-1modifier protein. BLAST protein searches can be performed with theXBLAST program, score=50, wordlength=3 to obtain amino acid sequenceshomologous to a SCA-1 modifier protein. To obtain gapped alignments forcomparison purposes, Gapped BLAST can be utilized as described inAltschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively,PSI-Blast can be used to perform an iterated search which detectsdistant relationships between molecules (Id.). When utilizing BLAST,Gapped BLAST, and PSI-Blast programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used. Seehttp://www.ncbi.nlm.nih.gov. Another preferred, non-limiting example ofa mathematical algorithm utilized for the comparison of sequences is thealgorithm of Myers and Miller, CABIOS (1989). Such an algorithm isincorporated into the ALIGN program (version 2.0) which is part of theGCG sequence alignment software package. When utilizing the ALIGNprogram for comparing amino acid sequences, a PAM120 weight residuetable, a gap length penalty of 12, and a gap penalty of 4 can be used.Additional algorithms for sequence analysis are known in the art andinclude ADVANCE and ADAM as described in Torellis and Robotti, 1994,Comput. Appl. Biosci., 10:3-5; and FASTA described in Pearson andLipman, 1988, Proc. Natl. Acad. Sci. 85:2444-8. Within FASTA, ktup is acontrol option that sets the sensitivity and speed of the search. Ifktup=2, similar regions in the two sequences being compared are found bylooking at pairs of aligned residues if ktup=1, single aligned aminoacids are examined. ktup can be set to 2 or 1 for protein sequences, orfrom 1 to 6 for DNA sequences. The default if ktup is not specified is 2for proteins and 6 for DNA. For a further description of FASTAparameters, seehttp://bioweb.pasteur.fr/docs/man/man/fasta.1.html#sect2, the contentsof which are incorporated herein by reference.

Alternatively, protein sequence alignment may be carried out using theCLUSTAL W algorithm, as described by Higgins et al., 1996, MethodsEnzymol. 266:383-402.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, only exact matches are counted.

5.4 Methods of Producing the Proteins of the Indention

The proteins, including antibodies, that are useful in the methods ofthe present invention can be produced by any method known in the art forthe synthesis of proteins, in particular, by chemical synthesis orpreferably, by recombinant expression techniques.

Recombinant expression of a protein that binds to CD30 and induces CD30signaling in a lymphocyte and/or exerts a cytotoxic or cytostatic effecton activated lymphocytes requires construction of an expression vectorcontaining a nucleic acid that encodes the protein. Once a nucleic acidencoding such a protein has been obtained, the vector for the productionof the protein molecule may be produced by recombinant DNA technologyusing techniques well known in the art. Thus, methods for preparing aprotein by expressing a nucleic acid containing nucleotide sequenceencoding said protein are described herein. Methods which are well knownto those skilled in the art can be used to construct expression vectorscontaining coding sequences and appropriate transcriptional andtranslational control signals. These methods include, for example, invitro recombinant DNA techniques, synthetic techniques, and in vivogenetic recombination. The invention, thus, provides replicable vectorscomprising a nucleotide sequence encoding a protein of the inventionoperably linked to a promoter. Wherein the protein is an antibody, thenucleotide sequence may encode a heave or light chain thereof, or aheave or light chain variable domain, operably linked to a promoter.Such vectors may include the nucleotide sequence encoding the constantregion of the antibody molecule (see, e.g., PCT Publication WO 86/05807;PCT Publication WO 89/01036; and U.S. Pat. No. 5,12′,464) and thevariable domain of the antibody may be cloned into such a vector forexpression of the entire heavy or light chain.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce a protein of the invention. Thus, the inventionencompasses host cells containing a nucleic acid encoding a protein ofthe invention, operably linked to a heterologous promoter. In preferredembodiments for the expression of double-chained antibodies, vectorsencoding both the heavy and light chains may be co-expressed in the hostcell for expression of the entire immunoglobulin molecule, as detailedbelow.

A variety of host-expression vector systems may be utilized to expressthe proteins molecules of the invention. Such host-expression systemsrepresent vehicles by which the coding sequences of interest may beproduced and subsequently purified, but also represent cells which may,when transformed or transfected with the appropriate nucleotide codingsequences, express a protein of the invention in situ. These include butare not limited to microorganisms such as bacteria (e.g., E. coli, B.subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA orcosmid DNA expression vectors containing antibody coding sequences;yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeastexpression vectors containing antibody coding sequences; insect cellsystems infected with recombinant virus expression vectors (e.g.,baculovirus) containing antibody coding sequences; plant cell systemsinfected with recombinant virus expression vectors (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed withrecombinant plasmid expression vectors (e.g., Ti plasmid) containingantibody coding sequences; or mammalian cell systems (e.g., COS, CHO,BHK, 293, 3T3 cells) harboring recombinant expression constructscontaining promoters derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g. the adenoviruslate promoter; the vaccinia virus 7.5K promoter). Preferably, bacterialcells such as Escherichia coli, and more preferably, eukaryotic cells,especially for the expression of whole recombinant antibody molecules,are used for the expression of a recombinant protein of the invention.For example, mammalian cells such as Chinese hamster ovary cells (CHO),in conjunction with a vector such as the major intermediate early genepromoter element from human cytomegalovirus is an effective expressionsystem for proteins of the invention (Foecking et al., 1986, Gene 45:101; Cockett et al., 1990, Bio/Technology 8:2).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the foldingand post-translation modification requirements protein being expressed.Where possible, when a large quantity of such a protein is to beproduced, for the generation of pharmaceutical compositions comprising aprotein of the invention, vectors which direct the expression of highlevels of fusion protein products that are readily purified may bedesirable. Such vectors include, but are not limited, to the E. coliexpression vector pUR278 (Ruther et al., 1983, EMBO 1. 2:1791), in whichthe antibody coding sequence may be ligated individually into the vectorin frame with the lac Z coding region so that a fusion protein isproduced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res.13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509);and the like. pGEX vectors may also be used to express fusion proteinswith glutathione S-transferase (GST). In general, such fusion proteinsare soluble and can easily be purified from lysed cells by adsorptionand binding to matrix glutathioneagarose beads followed by elution inthe presence of free glutatluone. The pGEX vectors are designed toinclude thrombin or factor Xa protease cleavage sites so that the clonedtarget gene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the coding sequence of the protein of the invention may beligated to an adenovirus transcription/translation control complex,e.g., the late promoter and tripartite leader sequence. This chimericgene may then be inserted in the adenovirus genome by in vitro or invivo recombination. Insertion in a non-essential region of the viralgenome (e.g., region E1 or E3) will result in a recombinant virus thatis viable and capable of expressing the protein of the invention ininfected hosts. (See, e.g., Logan & Shenk, 1984, Proc. Natl. Acad. Sci.USA 8 1:355-359). Specific initiation signals may also be required forefficient translation of inserted coding sequences. These signalsinclude the ATG initiation codon and adjacent sequences. Furthermore,the initiation codon must be in phase with the reading frame of thedesired coding sequence to ensure translation of the entire insert.These exogenous translational control signals and initiation codons canbe of a variety of origins, both natural and synthetic. The efficiencyof expression may be enhanced by the inclusion of appropriatetranscription enhancer elements, transcription terminators, etc. (seeBittner et al., 1987, Methods in Enzymol. 153:51-544).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein of the invention. Differenthost cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins and geneproducts. Appropriate cell lines or host systems can be chosen to ensurethe correct modification and processing of the foreign proteinexpressed. To this end, eukaryotic host cells which possess the cellularmachinery for proper processing of the primary transcript,glycosylation, and phosphorylation of the gene product may be used. Suchmammalian host cells include but are not limited to CHO, VERO, BHK,HeLa, COS, MDCK, 293, 3T3, and W138.

For long-term, high-yield production of recombinant proteins that bindto CD30 and exert a cytotoxic or cytostatic effect on activatedlymphocytes, stable expression is preferred. For example, cell lineswhich stably express the protein may be engineered. Rather than usingexpression vectors which contain viral origins of replication, hostcells can be transformed with DNA controlled by appropriate expressioncontrol elements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the proteinof the invention.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell11:223), hypoxanthineguanine phosphoribosyltransferase (Szybalska &Szybalski, 1992, Proc. Natl. Acad. Sci. USA 48:202), and adeninephosphoribosyltransferase (Lowy et al., 1980, Cell 22:8-17) genes can beemployed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., 1980, Proc. Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981,Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA78:2072); neo, which confers resistance to the aminoglycoside G-418(Goldspiel et al., Clinical Pharmacy 12:488-505; Wu and Wu, 1991,Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol.32:573-596; Mulligan, 1993, Science 260:926-932 and Morgan and Anderson,1993, Ann. Rev. Biochem. 62: 191-217; May, 1993, TIB TECH11(5):155-215); and hygro, which confers resistance to hygromycin(Santerre et al., 1984, Gene 30:147). Methods commonly known in the artof recombinant DNA technology may be routinely applied to select thedesired recombinant clone, and such methods are described, for example,in Ausubel et al. (eds.), Current Protocols in Molecular Biology, JohnWiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, ALaboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13,Dracopoli et al. (eds), Current Protocols in Human Genetics. John Wiley& Sons, NY (1994); Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1,which are incorporated by reference herein in their entireties.

The expression levels of a protein of the invention can be increased byvector amplification (for a review, see Bebbington and Hentschel, “TheUse of Vectors Based on Gene Amplification for the Expression of ClonedGenes in Mammalian Cells in DNA Cloning”, Vol.3. (Academic Press, NewYork. 1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe protein of the invention will also increase (Crouse et al., 1983,Mol. Cell. Biol. 3:257).

Wherein the protein of the invention is an antibody, the host cell maybe co-transfected with two expression vectors of the invention, thefirst vector encoding a heavy chain derived protein and the secondvector encoding a light chain derived protein. The two vectors maycontain identical selectable markers which enable equal expression ofheavy and light chain proteins. Alternatively, a single vector may beused which encodes, and is capable of expressing, both heavy and lightchain proteins. In such situations, the light chain should be placedbefore the heavy chain to avoid an excess of toxic free heavy chain(Proudfoot, 1986, Nature 322:52; Kohler, 1980, Proc. Natl. Acad. Sci.USA 77:2197). The coding sequences for the heavy and light chains maycomprise cDNA or genomic DNA.

Once a protein molecule of the invention has been produced by an animal,chemically synthesized, or recombinantly expressed, it may be purifiedby any method known in the art for purification of proteins, forexample, by chromatography (e.g., ion exchange; affinity, particularlyby affinity for the specific antigen, Protein A (for antibody molecules,or affinity for a heterologous fusion partner wherein the protein is afusion protein; and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins.

The present invention encompasses the use of proteins of the inventionthat are fusion proteins, i.e., proteins that are recombinantly fused orchemically conjugated (including both covalent and non-covalentconjugation) to heterologous proteins (of preferably at least 10, 20,30, 40, 50, 60, 70, 80, 90 or at least 100 amino acids). The fusion doesnot necessarily need to be direct, but may occur through linkersequences.

The present invention further includes compositions comprising proteinsof the invention fused or conjugated to antibody domains other than thevariable regions. For example, the proteins of the invention may befused or conjugated to an antibody Fc region, or portion thereof. Theantibody portion fused to a protein of the invention may comprise theconstant region, hinge region. CH 1 domain. CR2 domain, and CH3 domainor any combination of whole domains or portions thereof. The proteinsmay also be fused or conjugated to the above antibody portions to formmultimers. For example, Fc portions fused to the proteins of theinvention can form dimers through disulfide bonding between the Fcportions. Higher multimeric forms can be made by fusing the proteins toportions of IgA and IgM. Methods for fusing or conjugating the proteinsof the invention to antibody portions are known in the art. See, e.g.,U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053; 5,447,851;5,112,946; EP 307,434: EP 367,166; PCT publications WO 96/04388; WO 91/06570; Ashkenazi et al. 1991, Proc. Nat. Acad. Sci. USA88:10535-10539; Zheng et al., 1995, J. Immunol. 154:5590-5600; and Vilet al., 1992, Proc. Natl. Acad. Sci. USA 89:11337-11341 (said referencesincorporated by reference in their entireties).

5.5 Conjugates and Fusion Proteins

As discussed, supra, the methods of the invention for treatment andprevention of immunological disorders encompass the use of proteins thatbind to CD30 and exert a cytostatic and/or cytotoxic effect on activatedlymphocytes, and that are further fused or conjugated to heterologousproteins or cytotoxic agents.

The present invention thus provides for methods of treatment orprevention of immunological disorders by administration of a protein ornucleic acid of the invention. In certain embodiments of the invention,a protein or nucleic acid of the invention may be chemically modified toimprove its cytotoxic and/or cytostatic properties. For example, aprotein of the invention can be administered as a conjugate.Particularly suitable moieties for conjugation to proteins of theinvention are chemotherapeutic agents, pro-drug converting enzymes,radioactive isotopes or compounds, or toxins.

In one embodiment, a protein of the invention is fused to a markersequence, such as a peptide, to facilitate purification. In preferredembodiments, the marker amino acid sequence is a hexa-histidine peptide,such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 EtonAvenue. Chatsworth, Calif., 91311), among others, many of which arecommercially available. As described in Gentz et al. 1989, Proc. Natl.Acad. Sci. USA 86:821-824, for instance, hexa-histidine provides forconvenient purification of the fusion protein. Other peptide tags usefulfor purification include, but are not limited to, the “HA” tag, whichcorresponds to an epitope derived from the influenza hemagglutininprotein (Wilson et al., 1984, Cell 37:767) and the “flag” tag. Suchfusion proteins can be generated by standard recombinant methods knownto those of skill in the art.

In another embodiment, the proteins of the invention are fused orconjugated to a therapeutic agent. For example, a protein of theinvention may be conjugated to a cytotoxic agent such as achemotherapeutic agent (see infra Section 5.6), a toxin (e.g. acytostatic or cytocidal agent), or a radionuclide (e.g., alpha-emitterssuch as, for example, ²¹²Bi, ²¹¹At, or beta-emitters such as, forexample, ¹³¹I, ⁹⁰Y, or ⁶⁷Cu). Examples of additional agents that areuseful for conjugating to the anti-CD30 molecules of the invention areprovided in Section 5.12.1, infra.

The conjugates of the invention used for enhancing the therapeuticeffect of the anti-CD30 antibodies that are useful in the methods of thepresent invention include non-classical therapeutic agents such astoxins. Such toxins include, but are not limited to, abrin, ricin A,pseudomonas exotoxin, or diphtheria toxin.

Alternatively, an antibody of the invention can be conjugated to asecond antibody to form an antibody heteroconjugate as described bySegal in U.S. Pat. No. 4,676,980, which is incorporated herein byreference in its entirety. Heteroconjugates useful for practicing thepresent invention comprise antibodies or antibody portions that bind toCD30 (including but not limited to antibodies that have the CDRs and/orheavy chains of the monoclonal antibodies Ki-2, Ki-4, Ki-5, Ki-7,Ber-H2, HRS-1, HRS-4, Ki-1, Ki-6, M67, Ki-3, M44, HeFi-1, and AC10) andantibody or a antibody portions that bind to a lymphocyte surfacereceptor or receptor complex, such as an immunoglobulin gene superfamilymember, a TNF receptor superfamily member, an integrin, a cytokinereceptor, a chemokine receptor, a major histocompatibility protein, alectin (C-type, S-type, or I-type), or a complement control protein.Examples of such molecules, and antibodies against such molecules thatcan be used to make heteroconjugates, are provided in Section 5.12.2,infra.

As discussed above, in certain embodiments of the invention, a proteinof the invention can be co-administered with a pro-drug convertingenzyme. The pro-drug converting enzyme can be expressed as a fusionprotein with or conjugated to a protein of the invention. Exemplarypro-drug converting enzymes are carboxypeptidase G2, beta-glucuronidase,penicillin-V-amidase, penicillin-G-amidase, β-lactamase, β-glucosidase,nitroreductase and carboxypeptidase A.

5.6 Antibody-Drug Conjugates

The present invention encompasses the use of anti-CD30 antibody-drugconjugates (anti-CD30 ADCs) for the treatment or prevention of animmunological disorder. The ADCs of the invention are tailored toproduce clinically beneficial cytotoxic or cytostatic effects onCD30-expressing cells when administered to a patient with an immunedisorder involving CD30-expressing cells, preferably when administeredalone but also in combination with other therapeutic agents.

Techniques for conjugating such drugs to proteins, and in particular toantibodies, are well known, see, e.g., Arnon et al, “MonoclonalAntibodies For Immunotargeting Of Drugs In Cancer Therapy”, inMonoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp.243-56 (Alan R. Liss, Inc., 1985); Hellstrom et al., “Antibodies ForDrug Delivery”, in Controlled Drug Delivery (2nd ed.), Robinson et al.(eds.), pp. 623-53 (Marcel Dekker, Inc., 1987); Thorpe, “AntibodyCarriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in MonoclonalAntibodies'84: Biological And Clinical Applications, Pinchera et al.(eds.), pp. 475-506 (1985); “Analysis, Results, And Future ProspectiveOf The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., 1982,Immunol. Rev. 62:119-58.

Because in many of the disease states that are encompassed by thetreatment methods of the present invention a significant amount ofsoluble CD30 is shed from the activated lymphocytes, it is preferablewhen using an anti-CD30 antibody that is conjugated to a drug (e.g. acytotoxic agent or an immunosuppressive agent) or prodrug convertingenzyme that the drug or prodrug converting enzyme is active in thevicinity of the activated lymphocytes rather than any place in the bodythat soluble CD30 may be found.

Two approaches may be taken to minimize drug activity outside theactivated lymphocytes that are targeted by the anti-CD30 antibodies ofthe invention: first, an antibody that binds to cell membrane but notsoluble CD30 may be used, so that the drug, including drug produced bythe actions of the prodrug converting enzyme, is concentrated at thecell surface of the activated lymphocyte. A more preferred approach forminimizing the activity of drugs bound to the antibodies of theinvention is to conjugate the drugs in a manner that would reduce theiractivity unless they are hydrolyzed or cleaved off the antibody. Suchmethods would employ attaching the drug to the antibodies with linkersthat are sensitive to the environment at the cell surface of theactivated lymphocyte (e.g., the activity of a protease that is presentat the cell surface of the activated lymphocyte) or to the environmentinside the activated lymphocyte the conjugate encounters when it istaken up by the activated lymphocyte (e.g., in the endosomal or, forexample by virtue of pH sensitivity or protease sensitivity, in thelysosomal environment).

In one embodiment, the linker is an acid-labile hydrazone or hydrazidegroup that is hydrolyzed in the lysosome (see, e.g., U.S. Pat. No.5,622,929) In alternative embodiments, drugs can be appended toanti-CD30 antibodies through other acid-labile linkers, such ascis-aconitic amides, orthoesters, acetals and ketals (Dubowchik andWalker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol.Chem. 264:14653-14661). Such linkers are relatively stable under neutralpH conditions, such as those in the blood, but are unstable at below pH5, the approximate pH of the lysosome.

In other embodiments, drugs are attached to the anti-CD30 antibodies ofthe invention using peptide spacers that are cleaved by intracellularproteases. Target enzymes include cathepsins B and D and plasmin, all ofwhich are known to hydrolyze dipeptide drug derivatives resulting in therelease of active drug inside target cells (Dubowchik and Walker, 1999,Pharm. Therapeutics 83:67-123). The advantage of using intracellularproteolytic drug release is that the drug is highly attenuated whenconjugated and the serum stabilities of the conjugates can beextraordinarily high.

In yet other embodiments, the linker is a malonate linker (Johnson etal., 1995. Anticancer Res. 15:1387-93), a maleimidobeiizoyl linker (Lauet al., 1995, Bioorg-Med-Chem. 3(10):1299-1304), or a 3′-N-amide analog(Lau et al, 1995, Bioorg-Med-Chem. 3(103:1305-12).

The drugs used for conjugation to the anti-CD30 antibodies of thepresent invention can include conventional chemotherapeutics, such asdoxorubicin, paclitaxel, melphalan, vinca alkaloids, methotrexate,mitomycin C, etoposide, and others. In addition, potent agents suchCC-1065 analogues, calichiamicin, maytansine, analogues of dolastatin10, rhizoxin, and palytoxin can be linked to the anti-CD30 antibodiesusing the conditionally stable linkers to form potent immunoconjugates.Examples of other suitable drugs for conjugation to the anti-CD3 0antibodies of the present invention are provided in Section 5.12.1,infra.

5.6.1 Linkers

As discussed above in Section 5.6, ADCs are generally made byconjugating a drug to an antibody through a linker. Thus, a majority ofthe ADCs of the present invention, which comprise an anti-CD30 antibodyand a high potency drug and/or an internalization-promoting drug,further comprise a linker. Any linker that is known in the art may beused in the ADCs of the present invention, e.g., bifunctional agents(such as dialdehydes or imidoesters) or branched hydrazone linkers (see,e.g., U.S. Pat. No. 5,824,805, which is incorporated by reference hereinin its entirety).

In certain, non-limiting, embodiments of the invention, the linkerregion between the drug moiety and the antibody moiety of the anti-CD30ADC is cleavable or hydrolyzable under certain conditions, whereincleavage or hydrolysis of the linker releases the drug moiety from theantibody moiety. Preferably, the linker is sensitive to cleavage orhydrolysis under intracellular conditions.

In a preferred embodiment, the linker region between the drug moiety andthe antibody moiety of the anti-CD30 ADC is hydrolyzable if the pHchanges by a certain value or exceeds a certain value. In a particularlypreferred embodiment of the invention, the linker is hydrolyzable in themilieu of the lysosome, e.g., under acidic conditions (i.e., a pH ofaround 5-5.5 or less). In other embodiments, the linker is a peptidyllinker that is cleaved by a peptidase or protease enzyme, including butnot limited to a lysosomal protease enzyme, a membrane-associatedprotease, an intracellular protease, or an endosomal protease.Preferably, the linker is at least two amino acids long, more preferablyat least three amino acids long. Peptidyl linkers that are cleavable byenzymes that are present in CD30-expressing cancers are preferred. Forexample, a peptidyl linker that is cleavable by cathepsin-B (e.g., aGly-Phe-Leu-Gly linker), a thiol-dependent protease that is highlyexpressed in cancerous tissue, can be used. Other such linkers aredescribed, e.g., in U.S. Pat. No. 6,214,345, which is incorporated byreference in its entirety herein.

In other, non-mutually exclusive embodiments of the invention, thelinker by which the anti-CD30 antibody and the drug of an ADC of theinvention are conjugated promotes cellular internalization. In certainembodiments, the linker-drug moiety of the ADC promotes cellularinternalization. In certain embodiments, the linker is chosen such thatthe structure of the entire ADC promotes cellular internalization.

In a specific embodiment of the invention, derivatives ofvaline-citrulline are used as linker (val-cit linker). The synthesis ofdoxorubicin with the val-cit linker have been previously described (U.S.Pat. No. 6,214,345 to Dubowchik and Firestone, which is incorporated byreference herein in its entirety).

In another specific embodiment, the linker is a phe-lys linker.

In another specific embodiment, the linker is a thioether linker (see,e.g., U.S. Pat. No. 5,622,929 to Willner et al., which is incorporatedby reference herein in its entirety).

In yet another specific embodiment, the linker is a hydrazone linker(see, e.g., U.S. Pat. Nos. 5,122,368 to Greenfield et al. and 5,824,805to King et al., which are incorporated by reference herein in theirentireties).

In yet other specific embodiments, the linker is a disulfide linker. Avariety of disulfide linkers are known in the art, including but notlimited to those that can be formed using SATA(N-succinimidyl-S-acetylthioacetate), SPDP(N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB(N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT(N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene).SPDB and SMPT (see, e.g., Thorpe et al., 1987, Cancer Res.,47:5924-5931; Wawrzynczak et al., 1987, In Immunoconjugates: AntibodyConjugates in Radioimagery and Therapy of Cancer, ed. C. W. Vogel,Oxford U. Press, pp. 28-55; see also U.S. Pat. No. 4,880,935 to Thorpeet al., which is incorporated by reference herein in its entirety).

A variety of linkers that can be used with the compositions and methodsof the present invention are described in U.S. provisional applicationNo. 60/400,403, entitled “Drug Conjugates and their use for treatingcancer, an autoimmune disease or an infectious disease”, by Inventors:Peter D. Senter, Svetlana Doronina and Brian E. Toki, submitted on Jul.31, 2002, which is incorporated by reference in its entirety herein.

In yet other embodiments of the present invention, the linker unit of ananti-CD30 antibody-linker-drug conjugate (anti-CD30 ADC) links thecytotoxic or cytostatic agent (drug unit; -D) and the anti-CD30 antibodyunit (-A). As used herein the term anti-CD30 ADC encompasses anti-CD30antibody drug conjugates with and without a linker unit. The linker unithas the general formula:-T_(a)-W_(w)—Y_(y)—

-   -   wherein:

-T- is a stretcher unit;

-   -   a is 0 or 1;    -   each —W— is independently an amino acid unit;    -   w is independently an integer ranging from 2 to 12;    -   —Y— is a spacer unit; and    -   y is 0, 1 or 2.

5.6.1.1 The Stretcher Unit

The stretcher unit (-T-), when present, links the anti-CD30 antibodyunit to an amino acid unit (—W—). Useful functional groups that can bepresent on an anti-CD30 antibody, either naturally or via chemicalmanipulation include, but are not limited to, sulfhydryl, amino,hydroxyl, the anomeric hydroxyl group of a carbohydrate, and carboxyl.Preferred functional groups are sulfhydryl and amino. Sulfhydryl groupscan be generated by reduction of the intramolecular disulfide bonds ofan anti-CD30 antibody. Alternatively, sulfhydryl groups can be generatedby reaction of an amino group of a lysine moiety of an anti-CD30antibody with 2-iminothiolane (Traut's reagent) or other sulfhydrylgenerating reagents. In specific embodiments, the anti-CD30 antibody isa recombinant antibody and is engineered to carry one or more lysines.In other embodiments, the recombinant anti-CD30 antibody is engineeredto carry additional sulfhydryl groups, e.g., additional cysteines.

In certain specific embodiments, the stretcher unit forms a bond with asulfur atom of the anti-CD30 antibody unit. The sulfur atom can bederived from a sulfhydryl (—SH) group of a reduced anti-CD30 antibody(A). Representative stretcher units of these embodiments are depictedwithin the square brackets of Formulas (Ia) and (Ib; see infra), whereinA-, —W—, —Y—, -D, w and y are as defined above and R¹ is selected from—C₁-C₁₀ alkylene-, —C₃-C₈ carbocyclo-, —O—(C₁-C₈alkyl)-, -arylene-,—C₁-C₁₀ alkylene-arylene-, -arylene-C₁-C₁₀ alkylene-, —C₁-C₁₀alkylene-(C₃-C₈ carbocyclo)-, —(C₃-C₈ carbocyclo)-C₁-C₁₀ alkylene-,—C₃-C₈ heterocyclo-, C₁-C₁₀alkylene-(C₃-C₈ heterocyclo)-, —(C₃-C₈heterocyclo)-C₁-C₁₀ alkylene-, —(CH₂CH₂O)_(r)—, and —(CH₂CH₂O)_(r)—CH₂—;and r is an integer ranging from 1-10.

An illustrative stretcher unit is that of formula (Ia) where R¹ is—(CH₂)₅—:

Another illustrative stretcher unit is that of formula (Ia) where R¹ is—(CH₂CH₂O)_(r)—CH₂—: and r is 2:

Still another illustrative stretcher unit is that of formula (Ib) whereR¹ is —(CH₂)₅—:

In certain other specific embodiments, the stretcher unit is linked tothe anti-CD30 antibody unit (A) via a disulfide bond between a sulfuratom of the anti-CD30 antibody unit and a sulfur atom of the stretcherunit. A representative stretcher unit of this embodiment is depictedwithin the square brackets of Formula (II), wherein R¹, A-, —W—, —Y—,-D, w and y are as defined above

In even other specific embodiments, the reactive group of the stretchercontains a reactive site that can be reactive to an amino group of ananti-CD30 antibody. The amino group can be that of an arginine or alysine. Suitable amine reactive sites include, but are not limited to,activated esters such as succinimide esters, 4-nitrophenyl esters,pentafluorophenyl esters, anhydrides, acid chlorides, sulfonylchlorides, isocyanates and isothiocyanates. Representative stretcherunits of these embodiments are depicted within the square brackets ofFormulas (IIIa) and (IIIb), wherein R¹, A-, —W—, —Y—, -D, w and y are asdefined above;

In yet another aspect of the invention, the reactive function of thestretcher contains a reactive site that is reactive to a modifiedcarbohydrate group that can be present on an anti-CD30 antibody. In aspecific embodiment, the anti-CD30 antibody is glycosylatedenzymatically to provide a carbohydrate moiety. The carbohydrate may bemildly oxidized with a reagent such as sodium periodate and theresulting carbonyl unit of the oxidized carbohydrate can be condensedwith a stretcher that contains a functionality such as a hydrazide, anoxime, a reactive amine, a hydrazine, a thiosemicarbazone, a hydrazinecarboxylate, and an arylhydrazide such as those described by Kaneko, T.et al. Bioconjugate Chem 1991, 2, 133-41. Representative stretcher unitsof this embodiment are depicted within the square brackets of Formulas(IVa)-(IVc), wherein R¹, A-, —W—, —Y—, -D, xv and y are as definedabove.

5.6.1.2 The Amino Acid Unit

The amino acid unit (—W—) links the stretcher unit (-T-) to the Spacerunit (—Y—) if the Spacer unit is present, and links the stretcher unitto the cytotoxic or cytostatic agent (Drug unit; D) if the spacer unitis absent.

—W_(w)— is a dipeptide, tripeptide, tetrapeptide, pentapeptide,hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide,undecapeptide or dodecapeptide unit. Each —W— unit independently has theformula denoted below in the square brackets, and w is an integerranging from 2 to 12:

wherein R² is hydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl,p-hydroxybenzyl, —CH₂OH, —CH(OH)CH₃, —CH₂CH₂SCH₃, —CH₂CONH₂, —CH₂COOH,—CH₂CH₂CONH₂, —CH₂CH₂COOH, —(CH₂)₃NHC(═NH)NH₂, —(CH₂)₃NH₂,—(CH₂)₃NHCOCH₃, —(CH₂)₃NHCHO, —(CH₂)₄NHC(═NH)NH₂, —(CH₂)₄NH₂,—(CH₂)₄NHCOCH₃, —(CH₂)₄NHCHO, —(CH₂)₃NHCONH₂, —(CH₂)₄NHCONH₂,—CH₂CH₂CH(OH)CH₂NH₂, 2-pyridylmethyl-, 3-pyridylmethyl-,4-pyridylmethyl-, phenyl, cyclohexyl,

The amino acid unit of the linker unit can be enzymatically cleaved byan enzyme including, but not limited to, a tumor-associated protease toliberate the drug unit (-D) which is protonated in vivo upon release toprovide a cytotoxic drug (D).

Illustrative W_(w) units are represented by formulas (V)-(VII):

wherein R³ and R⁴ are as follows: R³ R⁴ benzyl (CH₂)₄NH₂; methyl(CH₂)₄NH₂; isopropyl (CH₂)₄NH₂; isopropyl (CH₂)₃NHCONH₂; benzyl(CH₂)₃NHCONH₂; isobutyl (CH₂)₃NHCONH₂; sec-butyl (CH₂)₃NHCONH₂;

(CH₂)₃NHCONH₂; benzyl methyl; and benzyl (CH₂)₃NHC(═NH)NH₂;

wherein R³, R⁴ and R⁵ are as follows: R³ R⁴ R⁵ benzyl benzyl (CH₂)₄NH₂;isopropyl benzyl (CH₂)₄NH₂; and H benzyl (CH₂)₄NH₂;

wherein R³, R⁴, R⁵ and R⁶ are as follows: R³ R⁴ R⁵ R⁶ H benzyl isobutylH; and methyl isobutyl methyl isobutyl.

Preferred amino acid units include, but are not limited to, units offormula (V) where: R³ is benzyl and R⁴ is —(CH₂)₄NH₂; R³ is isopropyland R⁴ is —(CH₂)₄NH₂; R³ is isopropyl and R⁴ is —(CH₂)₃NHCONH₂. Anotherpreferred amino acid unit is a unit of formula (VI), where: R³ isbenzyl, R⁴ is benzyl, and R⁵ is —(CH₂)₄NH₂.

—W_(w)— units useful in the present invention can be designed andoptimized in their selectivity for enzymatic cleavage by a particulartumor-associated protease. The preferred —W_(w)— units are those whosecleavage is catalyzed by the proteases, cathepsin B, C and D, andplasmin.

In one embodiment, —W_(w)— is a dipeptide, tripeptide or tetrapeptideunit.

Where R², R³, R⁴, R⁵ or R⁶ is other than hydrogen, the carbon atom towhich R², R³, R⁴, R⁵ or R⁶ is attached is chiral.

Each carbon atom to which R², R³, R⁴, R⁵ or R⁶ is attached isindependently in the (S) or (R) configuration.

In a preferred embodiment, the amino acid unit is a phenylalanine-lysinedipeptide (phe-lys or FK linker). In another preferred embodiment, theamino acid unit is a valine-citrulline dipeptide (val-cit or VC linker).

5.6.1.3 The Spacer Unit

The spacer unit (—Y—), when present, links an amino acid unit to thedrug unit. Spacer units are of two general types: self-immolative andnon self-immolative. A non self-immolative spacer unit is one in whichpart or all of the spacer unit remains bound to the drug unit afterenzymatic cleavage of an amino acid unit from the anti-CD30antibody-linker-drug conjugate or the drug-linker compound. Examples ofa non self-immolative spacer unit include, but are not limited to a(glycine-glycine) spacer unit and a glycine spacer unit (both depictedin Scheme 1). When an anti-CD30 antibody-linker-drug conjugate of theinvention containing a glycine-glycine spacer unit or a glycine spacerunit undergoes enzymatic cleavage via a tumor-cell associated-protease,a cancer-cell-associated protease or a lymphocyte-associated protease, aglycine-glycine-drug moiety or a glycine-drug moiety is cleaved fromA-T-W_(w)—. To liberate the drug, an independent hydrolysis reactionshould take place within the target cell to cleave the glycine-drug unitbond.

In a preferred embodiment, -Y_(y)— is a p-aminobenzyl ether which can besubstituted with Q_(m) where Q is is —C₁-C₈ alkyl, —C₁-C₈ alkoxy,-halogen, -nitro or -cyano; and m is an integer ranging from 0-4.

In one embodiment, a non self-immolative spacer unit (—Y—) is -Gly-Gly-.

In another embodiment, a non self-immolative the spacer unit (—Y—) is-Gly-.

In one embodiment, the drug-linker compound or an anti-CD30antibody-linker-dig conjugate lacks a spacer unit (y=0).

Alternatively, an anti-CD30 antibody-linker-drug conjugate of theinvention containing a self-immolative spacer unit can release the drug(D) without the need for a separate hydrolysis step. In theseembodiments, —Y— is a p-aminobenzyl alcohol (PAB) unit that is linked to—W_(w)— via the nitrogen atom of the PAB group, and connected directlyto -D via a carbonate, carbamate or ether group (Scheme 2 and Scheme 3).

where Q is —C₁-C₈ alkyl, —C₁-C₈ alkoxy, -halogen, -nitro or -cyano; m isan integer ranging from 0-4; and p is an integer ranging from 1-20.

where Q is —C₁-C₈ alkyl, —C₁-C₈ alkoxy, -halogen, -nitro or -cyano; m isan integer ranging from 0-4; and p is an integer ranging from 1-20.

Other examples of self-immolative spacers include, but are not limitedto, aromatic compounds that are electronically equivalent to the PABgroup such a 2-aminoimidazol-5-methanol derivatives (see Hay et al.,Bioorg. Med. Chem. Lett., 1999, 9, 2237 for examples) and ortho orpara-aminobenzylacetals. Spacers can be used that undergo facilecyclization upon amide bond hydrolysis, such as substituted andunsubstituted 4-aminobutyric acid amides (Rodrigues et al., Chemistry,Biology, 1995, 2, 223), appropriately substituted bicyclo[2.2.1] andbicyclo[2.2.2] ring systems (Storm, et al., J. Amer. Chem. Soc., 1972,94, 5815) and 2-aminophenylpropionic acid amides (Amsberry, et al., J.Org. Chem., 1990, 55, 5867). Elimination of amine-containing drugs thatare substituted at the α-position of glycine (Kingsbury, et al., J. Med.Chem., 1984, 27, 1447) are also examples of self-immolative spacerstrategies that can be applied to the anti-CD30 antibody-linker-drugconjugates of the invention.

In an alternate embodiment, the spacer unit is a branchedbis(hydroxymethyl)styrene (BHMS) unit (Scheme 4), which can be used toincorporate additional drugs.

where Q is —C₁-C₈ alkyl, —C₁-C₈ alkoxy, -halogen, -nitro or -cyano; m isan integer ranging from 0-4; n is 0 or 1; and p is an integer ragingfrom 1-20.

In one embodiment, the two -D moieties are the same.

In another embodiment, the two -D moieties are different.

Preferred spacer units (—Y_(y)—) are represented by Formulas (VIII)-(X):

where Q is C₁-C₈ alkyl, C₁-C₈ alkoxy, halogen. nitro or cyano; and m isan integer ranging from 0-4;

5.6.2 Drugs

The present invention encompasses the use of anti-CD30 ADCs for thetreatment or prevention of an immunological disorder. As used herein,the term “drug” or “cytotoxic agent,” where employed in the context ofan anti-CD30 ADC of the invention, does not include radioisotopes.Otherwise, any drug that is known to the skilled artisan can be used inconnection with the ADCs of the present invention.

The drugs used for conjugation to the anti-CD30 antibodies of thepresent invention can include conventional chemotherapeutics, such asdoxorubicin, paclitaxel, melphalan, vinca alkaloids, methotrexate,mitomycin C, etoposide, and others. In addition, potent agents suchCC-1065 analogues, calichiamicin, maytansine, analogues of dolastatin10, rhizoxin, and palytoxin can be linked to the anti-CD30 antibodiesusing the conditionally stable linkers to form potent immunoconjugates.Examples of other suitable drugs for conjugation to the anti-CD30antibodies of the present invention are provided in Section 5.12.1below.

In certain embodiments, the ADCs of the invention comprise drugs thatare at least 40-fold more potent than doxorubicin on CD30-expressingcells. Such drugs include, but are not limited to: DNA minor groovebinders, including enediynes and lexitropsins, duocarmycins, taxanes(including paclitaxel and docetaxel), puromycins, vinca alkaloids,CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin,cyanomorpholino-doxorubicin, echinomycin, combretastatin, netropsin,epithilone A and B, estramustine, cryptophysins, cemadotin,maytansinoids, dolastatins, e.g., auristatin E, dolastatin 10, MMAE,discodermolide, eleutherobin, and mitoxantrone.

In certain specific embodiments, an anti-CD30 ADC of the inventioncomprises an enediyne moiety. In a specific embodiment, the enediynemoiety is calicheamicin. Enediyne compounds cleave double stranded DNAby generating a diradical via Bergman cyclization.

A variety of cytotoxic and cytostatic agents that can be used with thecompositions and methods of the present invention are described in U.S.provisional application No. 60/400,403, entitled “Drug Conjugates andtheir use for treating cancer, an autoimmune disease or an infectiousdisease”, by Inventors: Peter D. Senter, Svetlana Doronina and Brian E.Toki, filed on Jul. 31, 2002, which is incorporated by reference in itsentirety herein.

In other specific embodiments, the cytotoxic or cytostatic agent isauristatin E or a derivative thereof.

In preferred embodiments, the auristatin E derivative is an ester formedbetween auristatin E and a keto acid. For example, auristatin E can bereacted with paraacetyl benzoic acid or benzoylvaleric acid to produceAEB and AEVB, respectively. Other preferred auristatin derivativesinclude MMAE and AEFP.

The synthesis and structure of auristatin E, also known in the art asdolastatin-10, and its derivatives are described in U.S. patentapplication Ser. Nos. 09/845,786 and 10/001,191; in the InternationalPatent Application No.: PCT/US02/13435, in U.S. Pat. Nos. 6,323,315;6,239,104; 6,034,065; 5,780,588; 5,665,860; 5,663,149; 5,635,483;5,599,902; 5,554,725; 5,530,097; 5,521,284; 5,504,191; 5,410,024;5,138,036; 5,076,973; 4,986,988; 4,978,744; 4,879,278; 4,816,444; and4,486,414, all of which are incorporated by reference in theirentireties herein.

In specific embodiments, the drug is a DNA minor groove binding agent.Examples of such compounds and their syntheses are disclosed in U.S.Pat. No. 6,130,237, which is incorporated by reference in its entiretyherein. In certain embodiments, the drug is a CBI compound.

In certain embodiments of the invention, an ADC of the inventioncomprises an anti-tubulin agent. Anti-tubulin agents are a wellestablished class of cancer therapy compounds. Examples of anti-tubulinagents include, but are not limited to, taxanes (e.g., Taxol®(paclitaxel), docetaxel), T67 (Tularik), vincas, and auristatins (e.g.,auristatin E, AEB, AEVB, MMAE, AEFP). Antitubulin agents included inthis class are also: vinca alkaloids, including vincristine andvinblastine, vindesine and vinorelbine; taxanes such as paclitaxel anddocetaxel and baccatin derivatives, epithilone A and B, nocodazole,colchicine and colcimid, estramustine, cryptophysins, cemadotin,maytansinoids, combretastatins, dolastatins, discodermolide andeleutherobin.

In a specific embodiment, the drug is a maytansinoid, a group ofanti-tubulin agents. In a more specific embodiment, the drug ismaytansine. Further, in a specific embodiment, the cytotoxic orcytostatic agent is DM-1 (ImmunoGen, Inc.; see also Chari et al. 1992,Cancer Res 52:127-131). Maytansine, a natural product, inhibits tubulinpolymerization resulting in a mitotic block and cell death. Thus, themechanism of action of maytansine appears to be similar to that ofvincristine and vinblastine. Maytansine, however, is about 200 to1,000-fold more cytotoxic in vitro than these vinca alkaloids.

In another specific embodiment, the drug is an AEFP.

In certain specific embodiments of the invention, the drug is not apolypeptide of greater than 50, 100 or 200 amino acids, for example atoxin. In a specific embodiment of the invention, the drug is not ricin.

In other specific embodiments of the invention, an ADC of the inventiondoes not comprise one or more of the cytotoxic or cytostatic agents thefollowing non-mutually exclusive classes of agents: alkylating agents,anthracyclines, antibiotics, antifolates, antimetabolites, antitubulinagents, auristatins, chemotherapy sensitizers, DNA minor groove binders,DNA replication inhibitors, duocarmycins, etoposides, fluorinatedpyrimidines, lexitropsins, nitrosoureas, platinols, purineantimetabolites, puromycins, radiation sensitizers, steroids, taxanes,topoisomerase inhibitors, vinca alkaloids, purine antagonists, anddihydrofolate reductase inhibitors. In more specific embodiments, thehigh potency drug is not one or more of an androgen, anthramycin (AMC),asparaginase, 5-azacytidine, azathioprine, bleomycin, busulfan,buthionine sulfoximine, camptothecin, carboplatin, carmustine (BSNU),CC-1065, chlorambucil, cisplatin, colchicine, cyclophosphamide,cytarabine, cytidine arabinoside, cytochalasin B, dacarbazine,dactinomycin (formerly actinomycin), daunorubicin, decarbazine,docetaxel, doxorubicin, an estrogen, 5-fluordeoxyuridine,5-fluorouracil, gramicidin D, hydroxyurea, idarubicin, ifosfamide,irinotecan, lomustine (CCNU), mechlorethamine, melphalan,6-mercaptopurine, methotrexate, mithramycin, mitomycin C, mitoxantrone,nitroimidazole, paclitaxel, plicamycin, procarbizine, streptozotocin,tenoposide, 6-thioguanine, thioTEPA, topotecan, vinblastine,vincristine, vinorelbine. VP-16, VM-26, azothioprine, mycophenolatemofetil, methotrexate, acyclovir, gangcyclovir, zidovudine, vidarabine,ribavarin, azidothymidine, cytidine arabinoside, amantadine,dideoxyuridine, iododeoxyuridine, poscarnet, and trifluridine.

5.6.2.1 Dolastatin Drugs

In certain embodiments, the cytotoxic or cytostatic agent is adolastatin. In more specific embodiments, the dolastatin is of theauristatin class. In a specific embodiment of the invention, thecytotoxic or cytostatic agent is MMAE (MMAE; Formula XI). In anotherspecific embodiment of the invention, the cytotoxic or cytostatic agentis AEFP (Formula XVI).

In certain embodiments of the invention, the cytotoxic or cytostaticagent is a dolastatin of formulas XII-XVIII.

5.6.3 Formation of Anti-CD30 Antibody-Drug Conjugates

The generation of anti-CD30 antibody drug conjugates (ADCs) can beaccomplished by any technique known to the skilled artisan. Briefly, theanti-CD30 ADCs comprise an anti-CD30 antibody, a drug, and a linker thatjoins the drug and the antibody. A number of different reactions areavailable for covalent attachment of drugs to antibodies. This is oftenaccomplished by reaction of the amino acid residues of the antibodymolecule, including the amine groups of lysine, the free carboxylic acidgroups of glutamic and aspartic acid, the sulfhydryl groups of cysteineand the various moieties of the aromatic amino acids. One of the mostcommonly used non-specific methods of covalent attachment is thecarbodiimide reaction to link a carboxy (or amino) group of a compoundto amino (or carboxy) groups of the antibody. Additionally, bifunctionalagents such as dialdehydes or imidoesters have been used to link theamino group of a compound to amino groups of the antibody molecule. Alsoavailable for attachment of drugs to antibodies is the Schiff basereaction. This method involves the periodate oxidation of a drug thatcontains glycol or hydroxy groups, thus forming an aldehyde which isthen reacted with the antibody molecule. Attachment occurs via formationof a Schiff base with amino groups of the antibody molecule.Isothiocyanates can also be used as coupling agents for covalentlyattaching drugs to antibodies. Other techniques known to the skilledartisan and within the scope of the present invention. Non-limitingexamples of such techniques are described in, e.g., U.S. Pat. Nos.5,665,358, 5,643,573, and 5,556,623, which are incorporated by referencein their entireties herein.

In certain embodiments, an intermediate, which is the precursor of thelinker, is reacted with the drug under appropriate conditions. Incertain embodiments, reactive groups are used on the drug and/or theintermediate. The product of the reaction between the drug and theintermediate, or the derivatized drug, is subsequently reacted with theanti-CD30 antibody under appropriate conditions. Care should be taken tomaintain the stability of the antibody under the conditions chosen forthe reaction between the derivatized drug and the antibody.

5.7 Assays for Cytotoxic and Cytostatic Activities

By definition, a protein of the invention must exert a cytostatic orcytotoxic effect on an activated lymphocyte. Activated lymphocytes thatcan be assayed for a cytostatic or cytotoxic effect of a CD30 bindingprotein may be cultured cell lines (e.g., Jurkat and CESS, both of whichare available from the ATCC; or Karpas 299 and L540, both of which areavailable from Deutsche Sammlung von Mikroorganismen und ZellkulturenGmbH), or from lymphocytes prepared from a fresh blood sample.Lymphocytes can be activated by the appropriate cocktails of antibodiesand cytokines, as will be recognized by one of skill in the art. Forexample, T lymphocytes can be activated using a combination anti-CD3 andanti-CD28 antibodies and IL-2, as described in Section 11 below.

Many methods of determining whether a protein exerts a cytostatic orcytotoxic effect on a cell are known to those of skill in the art, andcan be used to elucidate whether a particular protein is a protein ofthe invention. Illustrative examples of such methods are describedbelow.

Wherein a protein that binds to CD30 does not exert a cytostatic orcytotoxic effect on activated lymphocytes, the protein can bemultimerized according to the methods described in Section 5.1, supra,and the multimer assayed for its ability to exert a cytostatic orcytotoxic effect on activated lymphocytes.

In a preferred embodiment, the proteins of the invention arecross-linked prior to assessing their cytotoxic or cytostatic effect onactivated lymphocytes. In an exemplary embodiment, in which the proteinof the invention is an anti-CD30 antibody, the antibody can becross-linked in solution, and one or more dilutions of the anti-CD30antibody can be titrated into 96-well flat bottom tissue culture platesin the absence or presence of secondary antibodies. Activatedlymphocytes are then added to the plates at approximately 5,000cells/well. The cytostatic or cytotoxic effect can then be assessed asdescribed herein, for example as an inhibition of radiolabeled thymidineincorporation into the activated lymphocytes.

Once a protein is identified that both (i) binds to CD30 and (ii) exertsa cytostatic or cytotoxic effect on activated lymphocytes, itstherapeutic value is validated in an animal model. Exemplary animalmodels of immunological disorders are described in Section 5.7.1, infra.

In a preferred embodiment for determining whether an anti-CD30 antibodyexerts a cytostatic effect on activated lymphocytes, a thymidineincorporation assay may be used. For example, activated lymphocytes at adensity of 5,000 cells/well of a 96-well plated can be cultured for a72-hour period and exposed to 0.5 μCi of ³H-thymidine during the final 8hours of the 72-hour period, and the incorporation of ³H-thymidine intocells of the culture is measured in the presence and absence of theantibody.

There are many cytotoxicity assays known to those of skill in the art.Some of these assays measure necrosis, while others measure apoptosis(programmed cell death). Necrosis is accompanied by increasedpermeability of the plasma membrane; the cells swell and the plasmamembrane ruptures within minutes. On the other hand, apoptosis ischaracterized by membrane blebbing, condensation of cytoplasm and theactivation of endogenous endonucleases. Only one of these effects onactivated lymphocytes is sufficient to show that a CD30-binding proteinis useful in the treatment or prevention of activated lymphocytes as analternative to the assays measuring cytostatic or cytotoxic effectsdescribed above.

In one embodiment, necrosis measured by the ability or inability of acell to take up a dye such as neutral red, trypan blue, or ALAMAR™ blue(Page et al., 1993, Intl. J. of Oncology 3:473-476). In such an assay,the cells are incubated in media containing the dye, the cells arewashed, and the remaining dye, reflecting cellular uptake of the dye, ismeasured spectrophotometrically.

In another embodiment, the dye is sulforhodamine B (SRB), whose bindingto proteins can be used as a measure of cytotoxicity (Skehan et al.,1990, J. Nat'l Cancer Inst. 82:1 107-12).

In yet another embodiment, a tetrazolium salt, such as MTT, is used in aquantitative calorimetric assay for mammalian cell survival andproliferation by detecting living, but not dead, cells (see, e.g.,Mosmann, 1983, J. Immunol. Methods 65:55-63).

In yet another embodiment, apoptotic cells are measured in both theattached and “floating” compartments of the cultures. Both compartmentsare collected by removing the supernatant, trypsinizing the attachedcells, and combining both preparations following a centrifugation washstep (10 minutes, 2000 rpm). The protocol for treating tumor cellcultures with sulindac and related compounds to obtain a significantamount of apoptosis has been described in the literature (see, e.g.,Piazza et al., 1995, Cancer Research 55:3110-16). Features of thismethod include collecting both floating and attached cells,identification of the optimal treatment times and dose range forobserving apoptosis as detected by DNA fragmentation, and identificationof optimal cell culture conditions.

In yet another embodiment, apoptosis is quantitated by measuring DNAfragmentation. Commercial photometric methods for the quantitative invitro determination of DNA fragmentation are available. Examples of suchassays, including TUNEL (which detects incorporation of labelednucleotides in fragmented DNA) and ELISA-based assays, are described inBiochemical, 1999, no. 2, pp. 34-37 (Roche Molecular Biochemicals).

In yet another embodiment, apoptosis can be observed morphologically.Following treatment with a test protein or nucleic acid, cultures can beassayed for apoptosis and necrosis by fluorescence microscopy followinglabeling with acridine orange and ethidium bromide. The method formeasuring apoptotic cell number has previously been described by Duke &Cohen, 1992, Current Protocols In Immunology, Coligan et al., eds.,3.17.1-3.17.16. In another mode of the embodiment, cells can be labeledwith the DNA dye propidium iodide, and the cells observed formorphological changes such as chromatin condensation and marginationalong the inner nuclear membrane, cytoplasmic condensation, increasedmembrane blebbing and cellular shrinkage.

5.7.1 Animal Models of Immunological Disorders

The molecules of the invention can be tested or validated in animalmodels of immunological disorders before they are subjected to clinicaltesting. A number of established animal models of immunologicaldisorders are known to the skilled artisan, any of which can be used toassay the efficacy of the molecules of the invention. Non-limitingexamples of such models are described below.

Some examples for animal models of systemic and organ-specificautoimmune diseases including diabetes, lupus, systemic sclerosis,Sjögren's Syndrome, experimental autoimmune encephalomyelitis (multiplesclerosis), thyroiditis, myasthenia gravis, arthritis, uveitis,inflamatory bowel disease have been described by Bigazzi, P., “AnimalModels of Autoimmunity: Spontaneous and Induced”, in The AutoimmuneDiseases, Rose and Mackay (eds.), pp. 211-244 (Academic Press, 1998) andin “Animal Models for Autoimmune and Inflamatory, Disease”, in CurrentProtocols in Immunology, Coligan et al. (eds.), Chapter 15 (Wiley,1997).

Allergic conditions, e.g. asthma and dermatitis, can also be modeled inrodents. Airway hypersensitivity can be induced in mice by ovalbumin(Tomkinson et al., 2001, J. Immunol. 166:5792-5800) or Schistosomamansoni egg antigen (Tesciuba et al., 2001, J. Immunol. 167:1996-2003).The Nc/Nga strain of mice show marked increase in serum IgE andspontaneously develop atopic dermatitis-like leisons (Vestergaard etal., 2000, Mol. Med. Today 6:209-210; Watanabe et al., 1997, Int.Immunol. 9:461-466; Saskawa et al., 2001, Int. Arch. Allergy Immunol.126:239-247).

Injection of immuno-competent donor lymphocytes into a lethallyirradiated histo-incompatible host is a classical approach to induceacute GVHD in mice. Alternatively, the parent-B6D2F1 murine modelprovides a system to induce both acute and chronic GVHD. In this modelthe B6D2F1 mice are F1 progeny from a cross between the parental strainsof C57BL/6 and DBA/2 mice. Transfer of DBA/2 lymphoid cells intonon-irradiated B6D2F1 mice causes chronic GVHD, whereas transfer ofC57BL/6, C57BL/10 or B10.D2 lymphoid cells causes acute GVHD (Slaybacket al., 2000, Bone Marrow Transpl. 26: 931-938; Kataoka et al., 2001,Immunology 103:310-318).

Additionally, both human hematopoietic stem cells and mature peripheralblood lymphoid cells can be engrafted into SCID mice, and these humanlympho-hematopoietic cells remain functional in the SCID mice (McCune etal., 1988, Science 241:1632-1639; Kamel-Reid and Dick, 1988, Science24′:1706-1709; Mosier et al., 1988, Nature 335:256-259). This hasprovided a small animal model system for the direct testing of potentialtherapeutic agents on human lymphoid cells. For example, a human-mousechimera model has been applied to examine the therapeutic potentials ofanti-IL-4, anti-IL-13, anti-IL-5, and the double-mutein IL-4 (Tournoy etal., 2001, J. Immunol. 166:6982-6991).

5.8 Assays for Signaling Activity

In certain preferred embodiments, a protein of the invention is capableof inducing one or more hallmarks of signaling through CD30 upon bindingto a CD30-expressing lymphocyte. CD30-expressing lymphocytes that can beassayed for a signaling effect of a CD30 binding protein may be culturedcell lines (e.g., Jurkat and CESS, both of which are available from theATCC; or Karpas 299 and L540, both of which are available from DeutscheSammlung von Mikroorganismen und Zellkulturen GmbH), or lymphocytesprepared from a fresh blood sample.

In a preferred embodiment, the proteins of the invention arecross-linked prior to assessing their activity on activated lymphocytes.In an exemplary embodiment, where the protein of the invention is ananti-CD30 antibody, the anti-CD30 antibody can be cross-linked insolution. Briefly, one or more dilutions of the anti-CD30 antibody canbe titrated into 96-well flat bottom tissue culture plates in theabsence or presence of secondary antibodies. Lymphocytes are then addedto the plates at approximately 5,000 cells/well. The signaling activityof the antibody can then be assessed as described herein.

Many methods of determining whether a protein induces one or morehallmarks of signaling through CD30 are known to those of skill in theart. Illustrative examples of such methods are described below.

5.8.1 Calcium Release

In one embodiment, a protein of the invention can induce the release ofintracellular free Ca²⁺ in Jurkat cells when it is cross-linked, forexample with a secondary antibody. The release of intracellular freeCa²⁺ can be measured as described by Ellis et al. (1993, J. Immunol.,151, 2380-2389) or by Mond and Brunswick (1998, Current Protocols inImmunology, Unit 3.9, Wiley).

5.8.2 TRAF Localization

Four TNF receptor-associated factors (TRAFs) including TRAF1, TRAF2,TRAF3, and TRAF5 have been demonstrated to interact with the cytoplasmictail of CD30 (Gedrich et al., 1996, J. Biol. Chem., 271, 12852-12858;Lee et al., 1996. Proc. Natl. Acad. Sci. USA. 93, 9699-9703; Ansieau etal., 1996, Proc. Natl. Acad. Sci. USA., 93, 14053-14058; Aizawa et al.,1997, J. Biol. Chem. 272, 2042-2045; Tsitsikov et al., 1997, Proc. Natl.Acad. Sci. USA. 94, 1390-1395; Lee er al., 1997, J. Exp. Med., 185,1275-12S5; Duckett and Thompson, 1997. Genes Dev. 11, 2810-2821). Usingco-transfection studies, yeast two-hybrid screening, and GST fusionproteins, the TRAF interacting sites have been mapped to the carboxylterminal of the cytoplasmic tail of CD30, and the association betweenCD30 and the TRAFs in the cytosolic phase has been hypothesized to be akey event in the CD30-mediated signal cascade. The interaction betweenCD30 and TRAF does not appear to require CD30 ligation (Ansieau et al.,1996, Proc. Natl. Acad. Sci. USA., 93, 14053-14058; Aizawa et al., 1997,J. Biol. Chem., 272, 2042-2045). However, cross-linking of CD30 leads toa disappearance of TRAF1 and TRAF2 from the detergent-soluble fractionsof cell lysates (Duckett and Thompson, 1997, Genes Dev., 11, 2810-2821;Arch et al., 2000, Biochem. Biophys. Res. Commun., 272, 936-945). Thedisappearance of TRAF2 is accompanied by a corresponding increase in thequantity of TRAF2 detectable in the detergent-insoluble fractioncontaining the nuclei (Arch et al., 2000, Biochem. Biophys. Res.Commun., 272, 936-945). Further subcellular localization studies haveconfirmed that cross-linking of CD30 induces a translocation of TRAF2from the cytosol to the perinuclear region of cells (Arch et al., 2000,Biochem. Biophys. Res. Commun., 272, 936-945). Such CD30-mediatedtranslocation of TRAF2 is hypothesized to modulate cell survival byregulating the sensitivity of cells to undergo apoptosis induced byother TRAF-binding members of the TNF receptor superfamily (Duckett andThompson, 1997, Genes Dev., 11, 2810-2821; Arch et al., 2000, Biochem.Biophys. Res. Commun., 272, 936-945).

To determine whether an antibody of the invention induces nucleartranslocation of TRAF2, the antibody of the invention is contacted withCD30+ cells and a cross-linking agent, such as a secondary antibody.Confocal microscopy can then be used to compare localization of TRAF2 incells incubated with the antibody of the invention (plus cross-linkingreagent) versus cells not incubated with the antibody of the invention.

In an alternative embodiment, whether an antibody of the inventioninduces TRAF2 nuclear localization can be assayed by measuring theamount of TRAF2 in various cell fractions, for example on a WesternBlot. For example, 2 μg/ml of an antibody of the invention can beincubated with CD30⁺ cells at 0.5×10⁶/ml. The antibody is cross-linkedby 20 μg/ml of a secondary antibody (e.g., where the antibody of theinvention is a mouse monoclonal antibody, a goat anti-mouse IgG Fcspecific antibody (Jackson ImmunoReseach, West Grove, Pa.) can be usedas a secondary antibody) at 37° C. and 5% CO₂. At designated time-points(e.g., 2 to 24 hours), 5×10⁶ cells are removed and spun down. After twowashes with ice-cold PBS, cells are lysed at 100×10⁶/ml in a lysisbuffer (0.15 M NaCl, 0.05 M Tris-HCl, pH 8.0, 0.005 M EDTA, and 0.5%NP-40 or Triton X-100) supplemented with a protease inhibitor cocktail(Roche Diagnositc GmBH, Mannheim, Germany). Lysis is done at 4° C. for 2hours with constant mixing. After lysis, the detergent-soluble anddetergent-insoluble fractions are separated by centrifugation at14,000×g for 20 minutes. The detergent-soluble fraction is thentransferred to a separate tube and an equal volume of 2×SDS-PAGEreducing sample buffer is added to it. An equal volume of 1×SDS-PAGEreducing sample buffer is also added to the detergent-insolublefraction, i.e., the pellet after centrifugation. Both fractions areheated to 100° C. for 2 minutes. About 10 μl of the fractions from eachtime point is then resolved by 12% Tris-glycine SDS-PAGE (Invitrogen,Carlsbad, Calif.). Resolved proteins are Western-transferred onto PVDFmembranes (Invitrogen), which is blocked with Tris buffer saline (0.05 MTris-HCl, pH 8.0, 0.138 M NaCl, 0.0027 M KCl) supplemented with 0.05%Tween 20 and 5% BSA. The blots are immunoblotted with an anti-TRAF2antibody (Santa Cruz, San Diego, Calif.). The presence of TRAF2 proteinin the different fractions is detected by horseradish peroxidase(HRP)-conjugated F(ab′)₂ goat anti-rabbit IgG Fc (JacksonImmunoResearch) and the peroxidase substrate kit DAB (VectorLaboratories, Burlingame, Calif.). Alternatively, the SuperSignal WestPico Chemiluminescent Substrate kit (Pierce, Rockford, Ill.) can also beused for detection.

5.8.3 NF-κB Activation

Another well-defined signal transduction event that can be induced bycertain antibodies of the invention is the activation of NF-κB.Anti-CD30 mAbs including M44, M67, and Ber-H2 can activate NF-κB asdetected by standard mobility shift DNA-binding assay (McDonald et al.,1995, Eur. J. Immunol., 25, 2870-2876; Ansieau et al., 1996, Proc. Natl.Acad. Sci. USA. 93, 14053-14058: Horie et al., 1998, Int. Immunol., 10,203-210). Such effect can be observed in Hodgkin cells, T cells, andtransfectant expressing CD30 (McDonald et al., 1995, Eur. J. Immunol.,25, 2870-2876: Biswas et al., 1995, Immunity, 2, 587-596; Ansieau eral., 1996, Proc. Natl. Acad. Sci. USA., 93, 14053-14058; Horie et al.,1998, Int. Immunol., 10, 203-210). Initial mapping studies revealed thatthe interaction between TRAF1, TRAF2, and TRAF5 with the cytoplasmictail of CD30 was required for the CD30-mediated activation of NF-κB (Leeet al., 1996, Proc. Natl. Acad. Sci. USA., 93, 9699-9703; Ansieau etal., 1996, Proc. Natl. Acad. Sci. USA., 93, 14053-14058; Aizawa et al.,1997, J. Biol. Chem., 272, 2042-2045; Duckett et al., 1997, Mol. Cell.Biol., 17, 1535-1542). More recently, evidence has become available thatligation of CD30 by agonistic mAbs can also activate NF-κB via aTRAF2/5-independent pathway (Horie et al., 1998, Int. Immunol., 10,203-210). Some of the biological consequences of the CD30-mediatedactivation of NF-κB include activation of gene transcription (Biswas etal., 1995, Immunity, 2, 587-596; Maggi et al., 1995, Immunity, 3,251-255) and regulation of cell survival (Mir et al., 2000, Blood, 96,4307-4312; Horie etal., 2002, Oncogene, 21,2439-2503). Any of thesecharacteristics of NF-κB activation can be assayed to determine whetheran antibody of the invention induces one or more hallmarks of CD30signaling.

Whether NF-κB activation is induced in CD30⁺ cells by an antibody of theinvention can be measured by, for example, incubating CD30⁺ cells at3×10⁶/ml with the antibody at 2 μg/ml, the antibody then cross-linked(e.g., where the antibody is a mouse monoclonal antibody, the antibodycan be cross-linked by 20 μg/ml of a goat anti-mouse IgG Fc specificantibody (Jackson ImmunoReseach, West Grove, Pa.)) and the cultureincubated at 37° C. and 5% CO₂ for 1 hour with constant shaking. Thecell density is adjusted to 1.2×10⁶/ml, and incubation with shaking iscarried on for an additional hour. Thereafter, cell density is furtherreduced to 0.6×10⁶/ml, and cells are incubated for an additional 46hours at 37° C. and 5% CO₂ without any further shaking. At the end ofincubation, nuclear extracts can be prepared from stimulated cells andanalyzed for NT-κB activation.

NF-κB activation is assayed by collecting the cells by centrifugation at1850×g for 20 minutes and then washing them once in 5 packed cellvolumes of PBS. The cell pellet is resuspended in 5 packed cell volumesof a hypotonic buffer (0.01 M Hepes, pH 7.9, 0.0015 M MgCl₂, 0.01 M KCl,0.0002 M phenylmethyl sulphonyl fluoride, 0.0005 M dithiothreitol).Cells are collected by centrifugation at 1850×g for 5 minutes. Thepellet is then resuspended in 3 packed cell volumes of the hypotonicbuffer and allowed to swell on ice for 10 minutes. After that, swollencells are homogenized with slow up-and-down strokes in a Douncehomogenizer, using a tight B pestle. Cell lysis is monitored by trypanblue exclusion, and enough strokes should be applied to achieve morethan 80% cell lysis. The nuclei are pelleted by centrifugation at 3300×gfor 15 minutes. The supernatant (cytoplasmic extract) is removed. Thenuclear pellet is then resuspended in ½ packed nuclei volume of alow-salt buffer (0.02 M Hepes, pH 7.9, 25% volume/volume glycerol,0.0015 M MgCl₂, 0.02 M KCl, 0.0002 M EDTA, 0.0002 M phenylmethylsulphonyl fluoride, 0.0005 M dithiothreitol). An equal volume of ahigh-salt buffer (0.02 M Hepes, pH 7.9, 25% volume/volume glycerol,0.0015 M MgCl₂, 1.2 M KCl, 0.0002 M EDTA, 0.0002 M phenylmethylsulphonyl fluoride, 0.0005 M dithiothreitol) is then slowly added to thenuclei suspension with gentle stirring to give a final KCl concentrationof roughly 0.3 M. The extraction is allowed to continue for 30 minuteswith gentle stirring. After extraction, the nuclei are removed bycentrifugation at 25,000×g for 30 minutes. The nuclear extraction isthen dialyzed against 50 volumes of a dialysis buffer (0.02 M Hepes, pH7.9, 20% volume/volume glycerol, 0.1 M KCl, 0.0002 M EDTA, 0.0002 Mphenylmethyl sulphonyl fluoride, 0.0005 M dithiothreitol) until theconductivity of the nuclear extract is the same as the dialysis buffer.The nuclear extract is centrifuged once more at 25,000×g for 20 minutesto remove residual debris, and the protein concentration of thesupernatant is determined by the micro-BCA assay (Pierce).

The presence of NF-κB in nuclear extract of anti-CD30 stimulated cellscan be detected by standard mobility shift DNA-binding assay using theGel Shift Assay System (Promega, Madison, Wis.). A double strandedoligonucleotide probe containing a consensus NF-κB binding motif withthe sequence 5′-AGT TGA GGG GAC TTT CCC AGG C-3′ (SEQ ID NO:33) (Lenardoand Baltimore, 1989, Cell, 58, 227-229) is used as the specific probe todetect NF-κB in nuclear extracts. This probe is phosphorylated by T4polynucleotide kinase and [α-³²P]ATP. The phosphorylated probe ispurified by Sepharose G25 spin columns equilibrated with TE buffer (0.01M Tris-HCl, pH 8.0, 0.001 M EDTA). Purified probed is then precipitatedwith ammonium acetate and ethanol and then resuspended in 100 μl of TEbuffer. Reaction mixtures containing nuclear extracts fromanti-CD30-treated cells and control-treated cells are separatelycombined with the Gel Shift Binding buffer, water and unlabeledcompetitor probes according to the manufacturers instruction. Anunlabeled oligonucleotide containing the NF-κB consensus and anunlabeled irrelevant oligonucleotide are included in the reactionmixture as the sequence-specific and sequence-nonspecific competitors.After incubation for 10 minutes at room temperature, 1 μl of the³²P-labeled NF-κB consensus oligonucleotide is added to each reaction.The reactions are allowed to continue for an additional 20 minutes atroom temperature. At the end of the incubation, 1 μl of a 10× loadingbuffer (0.25M Tris-HCl, pH 7.5, 40% volume/volume glycerol, 0.2%bromophenol blue) is added to the reactions. The reactions are thenloaded into individual wells of a 6% DNA retardation gel (Invitrogen)and resolved at 100 volt for 90 minutes in 0.5×TBE (0.045M Tris-HCl,0.045 M boric acid, 0.001M EDTA). After electrophoresis, the gel iscovered with plastic wrap and exposed to X-ray film at −70° C. to detectthe specific interaction between NF-κB and the oligonucleotidecontaining the NF-κB binding sequence.

5.9 Immune Disorders

The methods of the present invention are useful for treating orpreventing an immunological disorder, wherein the immunological disorderis characterized by inappropriate activation of lymphocytes. As usedherein, the phrase “immunological disorder” does not encompassimmunological cancers such as Hodgkin's Disease and anaplastic largecell lymphoma. Treatment or prevention of an immunological disorder,according to the methods of the present invention, is achieved byadministering to a patient in need of such treatment or prevention aprotein, preferably an antibody, that binds to activated lymphocytesthat are associated with the disease state and exerts a cytotoxic orcytostatic effect on the lymphocytes.

Examples of diseases that can be treated or prevented by the methods ofthe present invention include, but are not limited to, rheumatoidarthritis, multiple sclerosis, endocrine ophthalmopathy, uveoretinitis,systemic lupus erythematosus, myasthenia gravis, Grave's disease,glomerulonephritis, autoimmune hepatological disorder, autoimmuneinflammatory bowel disease, anaphylaxis, allergic reaction, Sjogren'ssyndrome, juvenile onset (Type I) diabetes mellitus, primary biliarycirrhosis, Wegener's granulomatosis, fibromyalgia, inflammatory boweldisease, polymyositis, dermiatomyositis, multiple endocrine failure,Schmidt's syndrome, autoimmune uveitis, Addison's disease, adrenalitis,thyroiditis, Hashimoto's thyroiditis, autoimmune thyroid disease,pernicious anemia, gastric atrophy, chronic hepatitis, lupoid hepatitis,atherosclerosis, presenile dementia, demyelinating diseases, subacutecutaneous lupus erythematosus, hypoparathyroidism, Dressler's syndrome,autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura,hemolytic anemia, pemphigus vulgaris, pemphigus, dermatitisherpetiformis, alopecia arcata, pemphigoid, scleroderma, progressivesystemic sclerosis, CREST syndrome (calcinosis, Raynaud's phenomenon,esophageal dysmotility, sclerodactyly, and telangiectasia), adult onsetdiabetes mellitus (Type II diabetes), male and female autoimmuneinfertility, ankylosing spondolytis, ulcerative colitis, Crohn'sdisease, mixed connective tissue disease, polyarteritis nedosa, systemicnecrotizing vasculitis, juvenile onset rheumatoid arthritis, atopicdermatitis, atopic rhinitis, Goodpasture's syndrome, Chagas' disease,sarcoidosis, rheumatic fever, asthma, recurrent abortion,anti-phospholipid syndrome, farmer's lung, erythema multiforme, postcardiotomy syndrome, Cushing's syndrome, autoimmune chronic activehepatitis, bird-fancier's lung, allergic encephalomyelitis, toxicepidermal necrolysis, Alport's syndrome, alveolitis, allergicalveolitis, fibrosing alveolitis, interstitial lung disease, erythemanodosum, pyoderma gangrenosum, transfusion reaction, leprosy, malaria,leishmaniasis, trypanosomiasis, Takayasu's arteritis, polymyalgiarheumatica, temporal arteritis, schistosomiasis, giant cell arteritis,ascariasis, aspergillosis, Sampter's syndrome, eczema, lymphomatoidgranulomatosis, Behcet's disease, Caplan's syndrome, Kawasaki's disease,dengue, encephalomyelitis, endocarditis, endomyocardial fibrosis,endophthalmitis, erythema elevatum et diutinum, psoriasis,erythroblastosis fetalis, eosinophilic faciitis. Shulman's syndrome,Felty's syndrome, filariasis, cyclitis, chronic cyclitis, heterochroniccyclitis, Fuch's cyclitis, IgA nepliropathy, Henoch-Schonlein purpura,graft versus host disease, transplantation rejection, humanimmunodeficiency virus infection, echovirus infection, cardiomyopathy,Alzheimer's disease, parvovirus infection, rubella virus infection, postvaccination syndromes, congenital rubella infection, Eaton-Lambertsyndrome, relapsing polychondritis, cryoglobulinemia, Waldenstrom'smacroglobulemia, Epstein-Barr virus infection, mumps, Evan's syndrome,and autoimmune gonadal failure.

Accordingly, the methods of the present invention encompass treatment ofdisorders of B lymphocytes (e.g., systemic lupus erythematosus,Goodpasture's syndrome, rheumatoid arthritis, and type I diabetes),Th₁-lymphocytes (e.g., rheumatoid arthritis, multiple sclerosis,psoriasis, Sjorgren's syndrome, Hashimoto's thyroiditis, Grave'sdisease, primary biliary cirrhosis, Wegener's granulomatosis,tuberculosis, or acute graft versus host disease), and Th₂-lymphocytes(e.g., atopic dermatitis, systemic lupus erythematosus, atopic asthma,rhinoconjunctivitis, allergic rhinitis, Omenn's syndrome, systemicsclerosis, or chronic graft versus host disease).

An alternative way of classifying immunological disease states is by theunderlying biological mechanism. The present invention is directed totreatment and prevention of immunological diseases arising by any of thefollowing mechanisms, which are classified into four types:

Anaphylactic reactions. These reactions are mediated by IgE antibodieswhich bind to receptors on mast cells. When cross-linking occurs withantigens, the IgE antibodies stimulate the mast cells to release anumber of pharmacologically active substances that can cause thesymptoms characteristic of anaphylaxis. These reactions to antigenicchallenge are immediate and potentially life-threatening. Examples ofanaphylactic responses include, but are not limited to, allergicrhinitis, gastrointestinal allergy, atopic dermatitis, bronchial asthmaand equine heaves and laminitis.

Cytotoxic (cytolytic) reactions. These cell surface reactions resultfrom an interaction of antigen with IgM and/or IgG which activates thecomplement cascade, leading to the destruction of the cell. Examples ofcytolytic reactions include, but are not limited to, leukocytopenia,hemolytic disease of newborn and Goodpasture's disease. Autoimmunedisorders that involve cytotoxic/cytolytic reactions are hemolyticanemia, thrombocytopenia and thyroiditis.

Immune complex reactions. Immune complex reactions occur when largecomplexes of antigen and IgG or IgM accumulate in the circulation or intissue, fixing complement. Granulocytes are attracted to the site ofcomplement fixation and release damaging lytic enzymes from theirgranules. An example of this type of reaction is serum sickness.Autoimmune disorders that involve immune complex reactions includesystemic lupus erythematosus, chronic glomerulonephritis and rheumatoidarthritis.

Cell-mediated immunity (CMI) reaction, or delayed-type hypersensitivity(DTH). In contrast to the first three types of immune responses, thishypersensitivity response is mediated by T lymphocytes rather thanantibodies produced by B lymphocytes. Activated T lymphocytes releasecytokines which can result in the accumulation and activation ofmacrophages, K cells and NK cells, which cause local tissue damage. Thisreaction can occur 1-2 days after antigenic challenge.

5.10 Gene Therapy

In a specific embodiment, nucleic acids encoding proteins of theinvention are administered to treat, inhibit or prevent an immunologicaldisorder. Gene therapy refers to therapy performed by the administrationto a subject of an expressed or expressible nucleic acid. In thisembodiment of the invention, the nucleic acids produce their encodedprotein that mediates a therapeutic effect.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. Exemplary methods are describedbelow.

For general reviews of the methods of gene therapy, see, Goldspiel etal., 1993, Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596;Mulligan, 1993, Science 260:926-932; Morgan and Anderson, 1993, Ann.Rev. Biochem. 62:191-217; May, 1993, TIBTECH 1, 1(5):155-215. Methodscommonly known in the art of recombinant DNA technology which can beused are described in Ausubel et al. (eds.), Current Protocols inMolecular Biology, John Wiley & Sons, NY (1993); and Kriegler, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

In a preferred aspect, the therapeutic comprises nucleic acid sequencesencoding an antibody, said nucleic acid sequences being part ofexpression vectors that express the antibody or fragments or chimericproteins or have or light chains thereof in a suitable host. Inparticular, such nucleic acid sequences have promoters operably linkedto the antibody coding region, said promoter being inducible orconstitutive, and, optionally, tissue-specific. In another particularembodiment, nucleic acid molecules are used in which the antibody codingsequences and any other desired sequences are flanked by regions thatpromote homologous recombination at a desired site in the genome, thusproviding for intrachromosomal expression of the antibody encodingnucleic acids (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438. In specificembodiments, the expressed antibody molecule is a single chain antibody;alternatively, the nucleic acid sequences include sequences encodingboth the heavy and light chains, or fragments thereof, of the antibody.

Delivery of the nucleic acids into a patient may be either direct, inwhich case the patient is directly exposed to the nucleic acid ornucleic acid-carrying vectors, or indirect, in which case, cells arefirst transformed with the nucleic acids in vitro, then transplantedinto the patient. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid sequences are directlyadministered its vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, for example by constructing them as part of an appropriatenucleic acid expression vector and administering the vector so that thenucleic acid sequences become intracellular. Gene therapy vectors can beadministered by infection using defective or attenuated retrovirals orother viral vectors (see, e.g., U.S. Pat. No. 4,980,286); directinjection of naked DNA; use of microparticle bombardment (e.g., a genegun; Biolistic, Dupont); coating with lipids or cell-surface receptorsor transfecting agents; encapsulation in liposomes, microparticles, ormicrocapsules; administration in linkage to a peptide which is known toenter the nucleus; administration in linkage to a ligand subject toreceptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol.Chem. 262:4429-4432) (which can be used to target cell typesspecifically expressing the receptors); etc. In another embodiment,nucleic acid-ligand complexes can be formed in which the ligandcomprises a fusogenic viral peptide to disrupt endosomes, allowing thenucleic acid to avoid lysosomal degradation. In vet another embodiment,the nucleic acid can be targeted in vivo for cell specific uptake andexpression, by targeting a specific receptor (see, e.g., PCTPublications WO 92/06 180; WO 92/22635; WO92/20316; WO93/14188, and WO93/20221). Alternatively, the nucleic acid can be introducedintracellularly and incorporated within host cell DNA for expression byhomologous recombination (Koller and Smithies, 1989, Proc. Natl. Acad.Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438).

In a specific embodiment, viral vectors that contain nucleic acidsequences encoding an antibody of the invention are used. For example, aretroviral vector can be used (see Miller et al., 1993, Meth. Enzymol.217:581-599). These retroviral vectors contain the components necessaryfor the correct packaging of the viral genome and integration into thehost cell DNA. The nucleic acid sequences encoding the antibody to beused in gene therapy are cloned into one or more vectors, therebyfacilitating delivery of the gene into a patient. More detail aboutretroviral vectors can be found in Boesen et al., 1994, Biotherapy 6:291-302, which describes the use of a retroviral vector to deliver the mdr1 gene to hematopoietic stem cells in order to make the stem cells moreresistant to chemotherapy. Other references illustrating the use ofretroviral vectors in gene therapy are: Clowes et al., 1994, J. Clin.Invest. 93:644-651; Klein et al., 1994, Blood 83:1467-1473; Salmons andGunzberg, 1993, Human Gene Therapy 4:129-141; and Grossman and Wilson,1993, Curr. Opin. in Genetics and Devel. 3:110-114.

Another approach to gene therapy involves transferring a gene, e.g. anAC10 or HeFi-1 gene, to cells in tissue culture by such methods aselectroporation, lipofection, calcium phosphate mediated transfection,or viral infection. Usually, the method of transfer includes thetransfer of a selectable marker to the cells. The cells are then placedunder selection to isolate those cells that have taken up and areexpressing the transferred gene. Those cells are then delivered to apatient.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see, e.g., Loeffler and Behr, 1993, Meth.Enzymol. 217:599-618; Cohen et al., 1993, Meth. Enzymol. 217:618-644;Cline. 1985, Pharmac. Ther. 29:69-92) and may be used in accordance withthe present invention, provided that the necessary developmental andphysiological functions of the recipient cells are not disrupted. Thetechnique should provide for the stable transfer of the nucleic acid tothe cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a patient by variousmethods known in the art. Recombinant blood cells (e.g., hematopoieticstem or progenitor cells) are preferably administered intravenously. Theamount of cells envisioned for use depends on the desired effect,patient state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to fibroblasts; blood cells such as T lymphocytes, Blymphocytes, monocytes, macrophages, neutrophils, eosinophils,megakaryocytes, granulocytes; various stem or progenitor cells, inparticular hematopoietic stem or progenitor cells, e.g., as obtainedfrom bone marrow, umbilical cord blood, peripheral blood, fetal liver,etc.

In a preferred embodiment, the cell used for gene therapy is autologousto the patient.

In an embodiment in which recombinant cells are used in gene therapy,nucleic acid sequences encoding an antibody are introduced into thecells such that they are expressible by the cells or their progeny, andthe recombinant cells are then administered in vivo for therapeuticeffect. In a specific embodiment, stem or progenitor cells are used. Anystem and/or progenitor cells which can be isolated and maintained invitro can potentially be used in accordance with this embodiment of thepresent invention (see e.g. PCT Publication WO 94/08598; Stemple andAnderson, 1992, Cell 71:973-985; Rheinwald, 1980, Meth. Cell Bio.21A:229; and Pittelkow and Scott, 1986, Mayo Clinic Proc. 61:771).

In a specific embodiment, the nucleic acid to be introduced for purposesof gene therapy comprises an inducible promoter operably linked to thecoding region, such that expression of the nucleic acid is controllableby controlling the presence or absence of the appropriate inducer oftranscription.

The compounds or pharmaceutical compositions of the invention arepreferably tested in vitro, and then in vivo for the desired therapeuticor prophylactic activity, prior to use in humans. For example, in vitroassays to demonstrate the therapeutic or prophylactic utility of anprotein or pharmaceutical composition include determining the effect ofthe protein or pharmaceutical composition on an activated lymphocytes.The cytotoxic and/or cytostatic effect of the protein or composition onthe activated lymphocytes can be determined utilizing techniques knownto those of skill in the art. Alternatively, in vitro assays which canbe used to determine whether administration of a specific protein orpharmaceutical composition is indicated, include in vivo cell cultureassays in which activated lymphocytes, including activated lymphocytesfrom a patient, are grown in culture, and exposed to or otherwise aprotein or pharmaceutical composition, and the effect of such compoundupon the activated lymphocytes is observed.

5.11 Therapeutic/Prophylactic Administration and Compositions

The invention provides methods of treatment and prophylaxis byadministration to a subject of an effective amount of a CD30-bindingprotein which has a cytotoxic or cytostatic effect on activatedlymphocytes (i.e., a protein of the invention), a nucleic acid encodingsaid CD30-binding protein (i.e., a nucleic acid of the invention), or apharmaceutical composition comprising a protein or nucleic acid of theinvention (hereinafter, a pharmaceutical of the invention).

The outcome of the present therapeutic and prophylactic methods is to atleast produce in a patient a healthful benefit, which includes but isnot limited to: prolonging the lifespan of a patient, prolonging theonset of symptoms of an immune disorder, and/or alleviating a symptom ofthe immune disorder after onset of a symptom. Such a healthful benefitcan result inhibiting disease progression and/or reducing diseasesymptoms.

As used herein, the term “prevention” refers to administration of aprotein or nucleic acid of the invention to the patient before the onsetof symptoms or molecular indications of the immune disorder of interest,for example to an individual with a predisposition or at a high risk ofacquiring the immune disorder. In contrast, the term “treatment” refersto administration of a protein or nucleic acid of the present inventionto the patient after the onset of symptoms or molecular indications ofthe immune disorder at any clinical stage.

In a preferred embodiment, the protein of the invention is themonoclonal antibody AC10 or HeFi-1 or a fragment or derivative thereof.In a preferred aspect, a pharmaceutical of the invention comprises asubstantially purified protein or nucleic acid of the invention (e.g.,substantially free from substances that limit its effect or produceundesired side-effects).

The subject is preferably an animal, and is preferably a mammal,including but not limited to animals such as cows, pigs, horses,chickens, cats, dogs, etc. Most preferably, the subject is human.

Formulations and methods of administration that can be employed aredescribed above; additional appropriate formulations and routes ofadministration can be selected from among those described herein below.

Various delivery systems are known and can be used to administer anucleic acid or protein of the invention, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the compound, receptor-mediated endocytosis (see, e.g., Wuand Wu, 1987, J. Biol. Chem. 262:4429-4432), construction of a nucleicacid as part of a retroviral or other vector, etc. Methods ofintroduction include but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. Nucleic acids and proteins of the invention may beadministered by any convenient route, for example by infusion or bolusinjection, by absorption through epithelial or mucocutaneous linings(e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may beadministered together with other biologically active agents such aschemotherapeutic agents (see Section 5.2.1). Administration can besystemic or local.

In a specific embodiment, it may be desirable to administer the nucleicacid or protein of the invention by injection, bad means of a catheter,by means of a suppository, or by means of an implant, said implant beingof a porous, non-porous, or gelatinous material, including a membrane,such as a sialastic membrane, or a fiber. Preferably, when administeringa protein, including an antibody, of the invention, care must be takento use materials to which the protein does not absorb.

In another embodiment, the compound or composition can be delivered in avesicle, in particular a liposome (see Langer, 1990, Science249:1527-1533. Treat et al., 1989, in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss,New York, pp. 353-365; Lopez-Berestein, ibid. pp. 317-327; seegenerally, ibid.)

In yet another embodiment, the compound or composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton, 1989, CRC Crit. Ref. Biomed. Eng. 14:201;Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J.Med. 321:574). In another embodiment, polymeric materials can be used(see Medical Applications of Controlled Release, 1974, Langer and Wise(eds.), CRC Pres., Boca Raton, Fla.; Controlled Drug Bioavailability,Drug Product Design and Performance, 1984, Smolen and Ball (eds.),Wiley, New York; Ranger and Peppas, 1983, Macromol. Sci. Rev. Macromol.Chem. 23:61; see also Levy et al., 1985, Science 228:190; During et al.,1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105).

Other controlled release systems are discussed in the review by Langer,1990, Science 249:1527-1533.

In a specific embodiment where a nucleic acid of the invention isadministered, the nucleic acid can be administered in vivo to promoteexpression of its encoded protein, by constructing it as part of anappropriate nucleic acid expression vector and administering it so thatit becomes intracellular, e.g., by use of a retroviral vector (see U.S.Pat. No. 4,980,286), or by direct injection, or by use of microparticlebombardment (e.g., a gene gun; Biolistic, Dupont), or coating withlipids or cell-surface receptors or transfecting agents, or byadministering it in linkage to a homeobox-like peptide which is known toenter the nucleus (see e.g., Joliot et al., 1991, Proc. Natl. Acad. Sci.USA 88:1864-1868), etc. Alternatively, a nucleic acid can be introducedintracellularly and incorporated within host cell DNA for expression, byhomologous recombination.

As alluded to above, the present invention also provides pharmaceuticalcompositions (pharmaceuticals of the invention). Such compositionscomprise a therapeutically effective amount of a nucleic acid or proteinof the invention, and a pharmaceutically acceptable carrier. In aspecific embodiment, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly in humans. Theterm “carrier” refers to a diluent, adjuvant, excipient, or vehicle withwhich the therapeutic is administered. Such pharmaceutical carriers canbe sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water is a preferredcarrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike. The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like. The composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides. Oral formulationcan include standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, etc. Examples of suitable pharmaceutical carriersare described in “Remington's Pharmaceutical Sciences” by E. W. Martin.Such compositions will contain a therapeutically effective amount of thenucleic acid or protein of the invention, preferably in purified form,together with a suitable amount of carrier so as to provide the form forproper administration to the patient. The formulation should suit themode of administration.

In a preferred embodiment, the pharmaceutical of the invention isformulated in accordance with routine procedures as a pharmaceuticalcomposition adapted for intravenous administration to human beings.Typically, compositions for intravenous administration are solutions insterile isotonic aqueous buffer. Where necessary, the pharmaceutical ofthe invention may also include a solubilizing agent and a localanesthetic such as lignocaine to ease pain at the site of the injection.Generally, the ingredients are supplied either separately or mixedtogether in unit dosage form, for example, as a dry lyophilized powderor water free concentrate in a hermetically sealed container such as anampoule or sachette indicating the quantity of active agent. Where thepharmaceutical of the invention is to be administered by infusion, itcan be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the pharmaceutical of theinvention is administered by injection, an ampoule of sterile water forinjection or saline can be provided so that the ingredients may be mixedprior to administration.

The amount of the nucleic acid or protein of the invention which will beeffective in the treatment or prevention of an immunological disordercan be determined by standard clinical techniques. In addition, in vitroassays may optionally be employed to help identify optimal dosageranges. The precise dose to be employed in the formulation will alsodepend on the route of administration, and the stage of immunologicaldisorder, and should be decided according to the judgment of thepractitioner and each patient's circumstances. Effective doses may beextrapolated from dose-response curves derived from in vitro or animalmodel test systems.

5.12 Combination Therapy for Treatment of Immunological Disorders

The nucleic acids and proteins of the invention can be administeredtogether with one or more cytostatic, cytotoxic and/or immunosuppressiveagents for the treatment and prevention of immunological disorders.Additionally, combination therapy may include administration of an agentthat targets a receptor or receptor complex other than CD30 on thesurface of activated lymphocytes. An example of such an agent is asecond, non-CD30 antibody that binds to a molecule at the surface of anactivated lymphocyte. Another example is a ligand that targets such areceptor or receptor complex. Preferably, such an antibody or ligandbinds to a cell surface receptor on activated lymphocytes and enhancesthe cytotoxic or cytostatic effect of the anti-CD30 antibody bydelivering a cytostatic or cytotoxic signal to the activatedlymphocytes.

Such combinatorial administration can have an additive or synergisticeffect on disease parameters.

With respect to therapeutic regimens, in a specific embodiment., anucleic acid or protein of the invention is administered concurrentlywith an immunosuppressive agent or a molecule that targets a lymphocytecell surface receptor or receptor complex. In another specificembodiment, the immunosuppressive agent or lymphocyte cell surfacereceptor targeting-agent is administered prior or subsequent toadministration of a nucleic acid or protein of the invention, by atleast an hour and up to several months, for example at least an hour,five hours, 12 hours, a day, a week, a month, or three months, prior orsubsequent to administration of a nucleic acid or protein of theinvention.

5.12.1 Immunosuppressive, Cytotoxic and Cytostatic Agents

A useful class of immunosuppressive, cytotoxic or cytostatic agents forpracticing the combinatorial therapeutic regimens of the presentinvention include, but are not limited to, the following non-mutuallyexclusive classes of agents: alkylating agents, anthracyclines,antibiotics, antifolates, antimetabolites, antitubulin agents,auristatins, chemotherapy sensitizers, DNA minor groove binders, DNAreplication inhibitors, duocarmycins, etoposides, fluorinatedpyrimidines, lexitropsins, nitrosoureas, platinols, purineantimetabolites, puromycins, radiation sensitizers, steroids, taxanes,topoisomerase inhibitors, and vinca alkaloids.

Individual immunosuppressive, cytotoxic or cytostatic agents encompassedby the invention include but are not limited to an androgen, anthramycin(AMC), asparaginase, 5-azacytidine, azathioprine, bleomycin, busulfan,buthionine sulfoximine, camptothecin, carboplatin, carmustine (BSNU),CC-1065, chlorambucil, cisplatin, colchicine, cyclophosphamide,cytarabine, cytidine arabinoside, cytochalasin B, dacarbazine,dactinomycin (formerly actinomycin), daunorubicin, decarbazine,docetaxel, doxorubicin, an estrogen, 5-fluordeoxyuridine,5-fluorouracil, gramicidin D, hydroxyurea, idarubicin, ifosfamide,irinotecan, lomustine (CCNU), mechlorethamine, melphalan,6-mercaptopurine, methotrexate, mithramrycin, mitomycin C, mitoxantrone,nitroimidazole, paclitaxel plicamycin, procarbizine, streptozotocin,tenoposide, 6-thioguanine, thioTEPA, topotecan, vinblastine,vincristine, vinorelbine, VP-16 and VM-26.

In a preferred embodiment, the immunosuppressive, cytotoxic orcytostatic agent is an antimetabolite. The antimetabolite can be apurine antagonist (e.g. azothioprine) or mycophenolate mofetil), adihydrofolate reductase inhibitor (e.g., methotrexate), acyclovir,gangcyclovir, zidovudine, vidarabine, ribavarin, azidothymidine,cytidine arabinoside, amantadine, dideoxyuridine, iododeoxyuridine,poscarnet, and trifluridine.

In another preferred embodiment, the immunosuppressive, cytotoxic orcytostatic agent is tacrolimus, cyclosporine or rapamycin.

In another preferred embodiment, the immunosuppressive agent is aglucocorticoid or glucocorticoid analogue. Examples of glucocorticoidsuseful in the present methods include cortisol and aldosterone. Examplesof glucocorticoid analogues useful in the present methods includeprednisone and dexamethasone.

In yet another preferred embodiment, the immunosuppressive agent is ananti-inflammatory agent, such as consisting arylcarboxylic derivatives,pyrazole-containing derivatives, oxicam derivatives and nicotinic acidderivatives. Classes of anti-inflammatory agents useful in the methodsof the present invention include cyclooxygenase inhibitors,5-lipoxygenase inhibitors, and leukotriene receptor antagonists.

Suitable cyclooxygenase inhibitors include meclofenamic acid, mefenamicacid, carprofen, diclofenac, diflunisal, fenbufen, fenoprofen,ibuprofen, indomethacin, ketoprofen, nabumetone, naproxen, sulindac,tenoxicam, tolmetin, and acetylsalicylic acid.

Suitable lipoxygenase inhibitors include redox inhibitors (e.g.,catechol butane derivatives, nordihydroguaiaretic acid (NDGA),masoprocol, phenidone, lanopalen, indazolinones, naphazatrom,benzofuranol, alkylhydroxylamine), and non-redox inhibitors (e.g.,hydroxythiazoles, methoxyalkylthiazoles, benzopyrans and derivativesthereof, methoxytetrahydropyran, boswellic acids and acetylatedderivatives of boswellic acids, and quinolinemethoxyphenylacetic acidssubstituted with cycloalkyl radicals), and precursors of redoxinhibitors.

Other suitable lipoxygenase inhibitors include antioxidants (e.g.phenols, propyl gallate, flavonoids and/or naturally occurringsubstrates containing flavonoids, hydroxylated derivatives of theflavones, flavonol, dihydroquercetin, luteolin, galangin, orobol,derivatives of chalcone, 4,2°,4′-trihydroxychalcone, ortho-aminophenols,N-hydroxyureas, benzofuranols, ebselen and species that increase theactivity, of the reducing selenoenzymes), iron chelating agents (e.g.,hydroxamic acids and derivatives thereof. N-hydroxyureas,2-benzyl-1-naphthol, catechols, hydroxylamines, carnosol trolox C,catechol, naphthol, sulfasalazine, zyleuton, 5-hydroxyanthranilic acidand 4-(omega-arylalkyl)phenylalkanoic acids), imidazole-containingcompounds (e.g., ketoconazole and itraconazole), phenothiazines, andbenzopyran derivatives.

Yet other suitable lipoxygenase inhibitors include inhibitors ofeicosanioids (e.g., octadecatetraenoic, eicosatetraenoic,docosapentaenoic, eicosahexaenoic and docosahexaenoic acids and estersthereof, PGE1 (prostagiandin E1), PGA2 (prostaglandin A2), viprostol,15-monohydroxyeicosatetraenoic, 15-monohydroxy-eicosatrienoic and15-monohydroxyeicosapentaenoic acids, and leukotrienes B5, C5 and D5),compounds interfering with calcium flows, phenothiazines,diphenylbutylantines, verapamil, fuscoside, curcumin, chlorogenic acid,caffeic acid, 5,8,11,14-eicosatetrayenoic acid (ETYA),hydroxyphenylretinamide, Ionapalen, esculin, diethylcarbamazine,phenantroline, baicalein, proxicromil, thioethers, diallyl sulfide anddi-(1-propenyl) sulfide.

Leukotriene receptor antagonists include calcitriol, ontazolast, BayerBay-x-1005, Ciba-Geigy CGS-25019C, ebselen, Leo Denmark ETH-615, LillyLY-293111, Ono ONO-4057, Terumo TMK-6S8, Boehiringer IngleheimBI-RM-270, Lilly LY 213024, Lilly LY 264086, Lilly LY 292728, Ono ONOLB457, Pfizer 105696, Perdue Frederick PF 10042, Rhone-Poulenc Rorer RP66153, SmithKline Beecham SB-201146, SmithKline Beecham SB-201993,SmithKline Beecham SB-209247, Searle SC-53228, Sumitamo SM 15178,American Home Products WAY 121006, Bayer Bay-o-8276, Warner-LambertCI-987, Warner-Lambert CI-987BPC-15LY 223982, Lilly LY 233569, LillyLY-255283, MacroNex MNX-160, Merck and Co. MK-591, Merck and CO. MK-886,Ono ONO-LB-448, Purdue Frederick PF-5901, Rhone-Poulenc Rorer RG 14893,Rhone-Poulenc Rorer RP 66364, Rhone-Poulenc Rorer RP 69698, ShionoogiS-2474, Searle SC-41930, Searle SC-50505, Searle SC-51146, SearleSC-52798, SmithKline Beecham SK&F-104493, Leo Denmark SR-2566, TanabeT-757 and Teijin TEI-1338.

In certain preferred embodiments of the present invention, theimmunosuppressive, cytotoxic or cytostatic agent is conjugated to anantibody of the invention rather than being administered separately.Antibody-drug conjugates useful in the present methods are described inSection 5.6, supra.

5.12.2 Lymphocyte Receptor Targeting Agents

Agents that are particularly useful in the present combinatorial methodsare molecules that bind to lymphocyte cell surface, preferably against areceptor or receptor complex distinct from CD30. Besides CD30, a widevariety of receptors or receptor complexes expressed on lymphocytesurface are involved in regulating the proliferation, differentiation,and functions of different lymphocyte subsets. Such molecules can betargeted, for example, to provide additional cytostatic or cytotoxicsignals to activated lymphocytes.

In one embodiment, suitable receptors for targeting alongside CD30 areimmunoglobulin gene superfamily members, including but not limited toCD2, CD3, CD4, CD8, CD19, CD22, CD28, CD79, CD90, CD1152/CTLA-4, PD-1,and ICOS (Barclay et al., 1997, The Leucocyte Antigen FactsBook, 2nd ed,Academic Press; Coyle and Gurtierrez-Ramos, 2001, Nature Immunol.2:203-209). In another embodiment, TNF receptor superfamily members canbe targeted, including but not limited to CD27, CD40, CD95/Fas,CD134/OX40, CD137/4-1BB, TNF-R1, TNFR-2, RANK, TACI, BCMA,osteoprotegerin. Apo2/TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4, and APO-3.(Locksley et al., 2001, Cell, 104, 487-501). In yet another embodiment,an integrin can be targeted, including but not limited to CD11a, CD11b,CD11c, CD18, CD29, CD41, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f,CD103, and CD104 (Barclay et al., 1997, The Leucocyte Antigen FactsBook,2nd ed, Academic Press). In yet other embodiments, a suitable receptorfor targeting in addition to CD30 is a cytokine receptor (Fitzgerald etal., 2001, The Cytokine Factsbook. 2nd ed, Academic Press), a chemokinereceptor (Luther and Cyster, 2001, Nature Immunol. 2:102-107; Gerard andRollins. 2001, Nature Immunol. 2: 108-115), a major histocompatibilityprotein, a lectin (C-type, S-type, or I-type), or a complement controlprotein.

In one embodiment, agents that bind to these non-CD30 receptors orreceptor complexes enhance the cytotoxic or cytostatic effect of theCD30-binding agent (e.g., an anti-CD30 antibody) by delivering acytostatic or cytotoxic signal to the activated lymphocytes. Incombination with the anti-CD30 molecules of the invention, an additiveor synergistic effect on growth inhibition or apoptosis can be achievedin the targeted lymphocyte.

In another embodiment, agents against these receptors or receptorcomplexes need not be growth inhibitory or apoptotic on their own, but,in combination with a CD30-binding agent, an enhanced effect on growthinhibition or apoptosis beyond that induced by the CD30-binding agentalone can be achieved. In certain specific embodiments, the enhancedeffect is approximately a 5%, 10% 15%, 20%, 25%, 30%, 40%, 50%, 75%,100% or greater enhancement in the cytostatic or cytotoxic activity of agiven amount or concentration of a CD30-binding agent. In oneembodiment, the enhanced effect refers to an approximately 5%, 10% 15%,20%, 25%, 30%, 40%, 50%, 75% reduction in the ED₅₀ of the CD30-bindingagent, i.e., the amount of the CD30-binding agent capable of achievingthe same cytotoxic or cytostatic effect is less than what would berequired to achieve the same cytotoxic or cytostatic effect in theabsence of administration of such agents that bind to receptor orreceptor complexes other than CD30.

In one embodiment, targeting a non-CD30 receptor or receptor complexaccording to the methods of the present invention can be achieved byadministering a ligand.

In another embodiment, targeting can be achieved by administering anantibody against the receptor or receptor complex. The antibody can be apolyclonal antibody, a monoclonal antibody, an epitope-binding antibodyfragment, or another type of antibody derivative equivalent to thoseanti-CD30 derivatives described in Sections 5.1 and 5.4, supra. Incertain specific embodiments, the antibody is a multivalent antibody ora heteroconjugate comprising a CD30-binding portion, as described inSections 5.1 and 5.4.

A number of antibodies suitable for co-administration with anti-CD30 areknown in the art, as will be recognized by the skilled artisan. Listedbelow are exemplary, non-limiting examples of such antibodies: theanti-CD2 antibodies include BTI-322 (Medimmune) and UMCD2; the anti-CD3antibodies OKT3, “SMART” Anti-CD3 (Nuvion™; Protein Design Laboratories)FN18, UCHT1, 145-2C11, and HIT3a; the anti-CD5 antibodies HI211(6T-003). HISM2 (6T-004), MEM-128 (6T-014), 7.8 (6T-080, OKT1, UCHT2,and BL1a; the anti-CTLA-4 antibodies 11D4. 10AS, 7F8, 4F10,ANC152.2/8H5, and BNI3.1; and the anti-PD-1 antibody J43.

Natural ligands have also been defined for many of the receptors orreceptor complexes (Barclay et al., 1997, The Leucocyte AntigenFactsBook, 2nd ed, Academic Press; Coyle and Gurtierrez-Ramos, 2001,Nature Immunol. 2:203-20; Locksley et al., 2001, Cell 104:487-501).Listed below are exemplary, non-limiting examples of such ligands:LFA-3, a ligand for CD2; CD80 and CD86, ligands for CD28 and CTLA-4;PD-L1 and PD-L2, ligands for PD-1; B7RP-1, a ligand for ICOS; CD70, aligand for CD27; CD154, a ligand for CD40; FasL, a ligand for CD95/Fas;TNFa, a ligand for TNF-R1 and TNF-R2; TRANCE, a ligand for RANK, APRIL,a ligand for TACI; BLYS, a ligand for BCMA, TRAIL, a ligand forTRAIL-R1, -R2, —R3, and R4; and TWEAK, a ligand for APO-3.

5.13 Effective Dose

Toxicity and therapeutic efficacy of the proteins and compositions ofthe invention can be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., for determining the LD₅₀(the dose lethal to 0.50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index and itcan be expressed as the ratio LD₅₀/ED₅₀. Proteins that exhibit largetherapeutic indices are preferred. While proteins that exhibit toxicside effects may be used, care should be taken to design a deliverysystem that targets such proteins to the site of affected tissue inorder to minimize potential damage to uninfected cells and, thereby,reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch proteins lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound that achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

Generally, the dosage of a protein of the invention in a pharmaceuticalof the invention administered to a immunological disorders patient istypically 0.1 mg/kg to 100 mg/kg of the patient's body weight.Preferably, the dosage administered to a patient is between 0.1 mg/kgand 20 mg/kg of the patient's body weight, more preferably 1 mg/kg to 10mg/kg of the patient's body weight. Generally, human antibodies have alonger half-life within the human body than antibodies from otherspecies due to the immune response to the foreign proteins. Thus, lowerdosages of humanized, chimeric or human antibodies and less frequentadministration is often possible.

5.14 Formulations

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers or excipients. Pharmaceuticalcompositions comprising the anti-CD30 antibodies of the invention mayfurther comprise a second antibody, such as an antibody described inSection 5.12.2, supra, or an immunosuppressive agent, such as one ofthose enumerated in Section 5.12.1, supra.

Thus, the proteins and their physiologically acceptable salts andsolvates may be formulated for administration by inhalation orinsufflation (either through the mouth or the nose) or oral, buccal,parenteral or rectal administration.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate) lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate): or wetting agents (e.g., sodium lauryl sulphate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicles beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to givecontrolled release of the active compound.

For buccal administration the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the proteins for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, e.g., dichlorodifluoromethane,triclilorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The proteins may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multidose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The proteins may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the proteins mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, theproteins may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

The compositions may, if desired, be presented in a pack or dispenserdevice that may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration preferably foradministration to a human.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

The invention is further described in the following examples which arein no way intended to limit the scope of the invention.

6. EXAMPLE 1 Expression of CD30 on the Jurkat Human T Lymphocyte CellLine

The Jurkat cell line is an acute T leukemia cell line that expresses theCD3/T cell receptor (TCR) complex and other important accessorymolecules involved in T cell functions. Sub-lines derived from theoriginal Jurkat line have been applied extensively as model systems toelucidate signaling pathways mediated by a multitude of receptorsystems, e.g., CD3/T cell receptor (TCR). A number of signaling pathwaysin Jurkat T cells initiated upon the ligation of surface receptors havebeen demonstrated to take place in normal T lymphocytes subjected toantigenic challenge. Jurkat T cells were found to consistently expressdetectable levels of CD30 (FIG. 1), and therefore they may be a modelsystem to examine the function of CD30 in activated lymphocytes.

7. EXAMPLE 2 Cross-Linking of CD30 on Jurkat T Cells by Anti-CD30 mAbsInhibited DNA Synthesis

The effect of signaling through CD30 by anti-CD30 on the proliferationof Jurkat T cells was assessed by tritiated thymidine (³H-TdR)incorporation assays. Jurkat cells were treated with soluble anti-CD30in graded doses or anti-CD30 cross-linked by a secondary cross-linkingantibody (Ab). For secondary cross-linking of cAC10, the monoclonalantibody (mAb) was mixed with F(ab′)₂ fragments of goat anti-human (GAM)IgG Fc (Jackson ImmunoResearch, West Groove, Pa.) in culture medium(RPMI-1640, 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, and 0.1 mMnon-essential amino acids). For secondary cross-linking of murineanti-CD30 mAb (AC10 and HeFi-1), the mAbs were mixed with F(ab′)₂fragments of goat anti-mouse (GAM) IgG Fc (Jackson ImmunoResearch) inculture medium. Final ratios of 1:2.5, 1:5, and 1:10 between the primarymAbs and secondary cross-linking antibodies were used. Antibodycocktails were allowed to incubate at room temperature for 15 minutes.Serial dilutions of these antibody cocktails in culture medium were thenprepared to achieve the desired final concentrations. One hundred ill ofantibody cocktails were then mixed with 100 μl Jurkat cell suspensioncontaining 5000 cells in 96-well tissue culture (TC) plates. Cells wereincubated with the antibody cocktails for 44 hours before a 4-hour pulsewith 1 μCi of ³H-TdR. Cells were then harvested onto filters, andincorporated tritiated thymidine was measured by scintillation counting.Thymidine incorporation values obtained from the treated cells were thencompared to the untreated control cultures.

Chimeric AC10 (cAC10) without secondary cross-linking did not inhibitJurkat cell proliferation at concentrations below 1 μg/ml. Cross-linkingcAC10 wraith a secondary Ab lowered the concentration of cAC10 needed tosignificantly inhibit Jurkat cell proliferation. Maximal inhibition wasachieved at concentrations of cAC10 as little as 0.1 μg/ml (FIG. 2).Both AC10 and HeFi-1 inhibited thymidine incorporation in Jurkat celldose-dependently when a secondary cross-linking antibodies was used(FIG. 3). The use of cross-linking antibodies in vitro likely simulatescross-linking via binding of the Fc portion of antibody to Fc receptorsexpressed on monocyte, macrophages, B lymphocytes, and NK cells in vivo.A ratio of 1:10 between the primary and secondary cross-linking antibodyappeared to be most efficient for proliferation inhibition. Detectableinhibition was observed at primary antibody concentrations of more than0.001 μg/ml for both AC10 and HeFi-1. Maximal level of inhibition wasobserved at concentrations greater than 0.1 μg/ml. AC10 and HeFi-1immobilized onto TC wells were also active in inhibiting Jurkat T cellproliferation (data not shown).

8. EXAMPLE 3 Cross-Linking of CD30 by Anti-CD30 mAbs Induced DNAFragmentation (Apoptosis) in Jurkat T Cells

The relationship between cell cycle status and DNA replication in Jurkatcells treated with cross-linked anti-CD30 in Abs was furtherinvestigated. Four μg/ml of the anti-CD30 mAb AC10 or HeFi-1 were mixedwith F(ab′)₂ fragments of GAM IgG Fc (Jackson ImmunoResearch) at finalratios (weight:weight) of 1:4 in culture medium (RPMI-1640, 10% FBS, 2mM L-glutamine, 1 mM sodium pyruvate, and 0.1 mM non-essential aminoacids). The antibody cocktails were allowed to incubate at roomtemperature for 15 minutes. Serial 1:10 dilutions of these antibodycocktails in culture medium were then prepared. One ml of antibodycocktails was then mixed with 1 ml of Jurkat cell suspension containing0.25×10⁶ cells in 12-well TC plates.

After 24 or 48 hours of incubation, cells were harvested bycentrifugation and resuspended in 2 ml of fresh medium equilibrated at37° C. Bromodeoxyuridine (BrdU) (Sigma, St. Louis, Mo.) was used tolabel cells that were actively synthesizing DNA. Twenty μl of 1 mM BrdUdissolved in water were added to the cell suspension, and the incubationcontinued at 37° C. for 15 minutes. Cells were then spun down, washedonce with PBS, and resuspended in 0.5 ml of ice-cold PBS. One ml of 100%ethanol at −20° C. was added drop-wise to the cell suspension for cellfixation. After fixation at 4 C for 30 minutes, fixed cells were spundown. The cell pellets were resuspended in 1 ml of 2 M HCl with 0.5%Triton X-100 (Sigma) and incubated for 30 minutes at room temperaturebefore cells were spun down. One ml of 0.1 M Na₂B₄O₇ at pH 8.5 was usedto resuspend the cell pellets and to neutralize the acid for 10 minutesat room temperature. Cells were then pelleted by centrifugation andwashed once with PBS containing 0.5% Tween 20 (Sigma) and 1% BSA. Fiftyμl of PBS, 0.5% Tween 20, 1% BSA were used to resuspend each cellpellet, to which 25 μl of FITC labeled anti-BrdU (BD ImmunocytometrySystems, San Jose, Calif.) were added, and the cell suspension wereincubated at room temperature for 30 minutes. Cells were then washedtwice with PBS, 0.5% Tween 20, 1% BSA, and then resuspended in 0.5 ml ofPBS containing 5 μg/ml of propidium iodide (PI) (Sigma) to quantify DNAcontents in cells. DNA synthesis and cell cycle status were thenexamined by flow cytometry.

FIG. 4 shows the appearance of Jurkat cells with DNA fragmentation incultures treated with either AC10 or HeFi-1 cross-linked by a goatanti-mouse IgG Fc antibody. Cells with DNA fragmentation are representedby events showing less than G₀/G₁ phase DNA content (Window 1 in FIG. 4)and events showing less than G₂/M phase DNA content that did notincorporate BrdU, i.e., cells not actively synthesizing DNA, (Window 4in FIG. 4). DNA fragmentation was detectable after 24 and 48 hours ofincubation. Such DNA fragmentation in Jurkat cell is characteristics ofapoptosis. These data suggest that the inhibition of proliferation ofJurkat cells shown in FIG. 5 was accompanied by cells undergoingapoptosis in response to CD30 signaling. When the percentages of cellsin different parts of the cell cycle from cultures treated with gradeddoses of AC10 and HeFi-1 were compiled, it was evident that AC10 orHeFi-1 at concentrations of >0.002 μg/ml were able to induce substantialapoptosis in Jurkat cells, especially after 48 hours of treatment (FIG.5). The appearance of apoptotic cells was paralleled by a correspondingdecrease in the percentages of cells in the G₀/G₁, S, and G₂/M phases ofthe cell cycle (FIG. 5).

9. EXAMPLE 4 Co-Cross-Linking of CD3 with CD30 Enhanced ApoptosisInduced in Jurkat T Cells

Activation-induced cell death (ACID) is a key mechanism used by theimmune system to eliminate auto-reactive lymphocytes and effectorlymphocytes thereby conferring tolerance to self-antigens andterminating immune responses, respectively. Ligation of CD3/TCRcomplexes on T lymphocytes or the sIg/B cell antigen receptor complexeson B lymphocytes activates T and B lymphocytes. Depending on the contextof antigen receptor ligation and signaling through accessory receptors,lymphocytes can proliferate and differentiate into effector cells orthey can undergo apoptosis. A number of accessory receptors, e.g., CD19,CD27, CD28, CD40, CD134/OX40, CD137/4-1BB, and ICOS co-stimulate withthe antigen receptors on B or T lymphocytes to promote cellularproliferation and/or differentiation. Hence, they are important forpromoting immune responses. On the other hand, signaling throughaccessory receptors including CD5, CD22, CD152/CTLA4, and PD-1 inhibitslymphocyte proliferation and various lymphocyte functions. Therefore,these receptors may play important roles in the elimination ofauto-reactive lymphocytes. They are also involved in the termination ofimmune responses by suppressing the activities of effector lymphocytes.Defects in regulating signal transduction pathways mediated by accessoryreceptors may contribute to autoimmune, allergic, and inflammatorydiseases.

We asked the question if the CD30 signaling pathway cooperates with thatof the CD3/TCR complex to regulate apoptosis in lymphocytes. Jurkat Tcells were treated with either anti-CD30 mAb alone, anti-CD3 mAb, alone,or a combination of anti-CD30 and anti-CD3 mAbs. Antibodies werecross-linked by goat anti-mouse IgG Fc as described for FIG. 4 and FIG.5. Cell cycle distribution was determined by BrdU and PI staining as inFIG. 4. Anti-CD30 (AC10) alone induced apoptosis in Jurkat T cells,whereas anti-CD3 (OKT3, ATCC, Manassas, Va.) did not have any effectcompared to medium control. However, anti-CD3 greatly enhanced apoptosisinduced by anti-CD30 after 24 and 48 hours of incubation. The bestsynergistic effect between AC10 and OKT3 was observed when both mAbswere used at 0.2 μg/ml (FIG. 6).

Binding of Annexin V onto the extracellular face of the plasma membranecoupled with membrane permeability to PI was used as a complementaryapproach to study anti-CD30-induced apoptosis in Jurkat cells. Antibodycocktails containing AC10, HeFi-1. OKT3, AC10+OKT3, or HeFi-1+OKT3cross-linked with a GAM antibody were prepared as described in Examples2 and 3. Primary mAbs were used at a final concentration of 2 μg/ml. A10-fold excess of GA-M antibody was used to cross-link the primaryantibodies. After an incubation of 15 minutes at room temperature,antibody cocktails were added to Jurkat cells. At the times designatedin FIG. 7, percentages of apoptotic and dead cells in the cultures weredetermined by Annexin V binding and permeability to PI using the AnnexinV-FITC Apoptosis Detection Kit I (BD PharMingen, San Diego, Calif.)according to the manufacturer's instruction. Cells that were AnnexinV⁻/PI⁻ were live cells (lower left quadrants in FIG. 7), cells that wereAnnexin V⁺/PI⁻ were apoptotic cells (lower right quadrants in FIG. 7),and cells that were Annexin V⁺/P⁺ were dead cells (upper right quadrantsin FIG. 7). Consistent to the data obtained from PI/BrdU staining shownin FIG. 6 AC10 and HeFi-1 induced detectable apoptosis in Jurkat cellsas early as 4 hours after incubation. The presence of an anti-CD3 mAbsignificantly enhanced the effects of AC10 and HeFi-1. The percentagesof apoptotic and dead cells continued to increase during the 48 hours ofincubation. These data suggest that the specific combination of anti-CD3and anti-CD30 mAbs may have significant ability to delete CD30 positiveT lymphocytes for controlled suppression of the immune responses inautoimmunity, allergic reactions and chronic inflamatory diseases.

10. EXAMPLE 5 cAC10 Antibody Drug Conjugates (ADCs)

10.1 Synthesis of cAC10-vcMMAE

The synthesis of the ADC cAC10-vcMMAE, the general structure of which isdepicted above, is described below.

10.1.1 Drug-Linker Compound Synthesis

The synthesis of auristatin E has been previously described (Pettit G R,and Barkoczy, J., 1997, U.S. Pat. No. 5,635,483, Pettit, G R, TheDolastatins, Prog. Chem. Org. Nat. Prod., 70, 1-79, 199). The monomethylderivative of Auristatin E (MMAE) was prepared by replacing a protectedform of monomethylvaline for N,N-dimethylvaline in the synthesis ofauristatin E (Senter et al., U.S. provisional application No. 60/400,403filed Jul. 31, 2002, which is incorporated by reference herein in itsentirety).

To prepare drug-linker compound, MMAE (1.69 g, 2.35 mmol),maleimidocaproyl-L-valine-L-citrulline-p-aminobenzyl alcoholp-nitrophenylcarbonate (2.6 g, 3.52 mmol, 1.5 eq., prepared as describedin Dubowchik. G. M., et al., Bioconjugate Chem. 2002, 13, 855-869) andHOBt (64 mg, 0.45 mmol, 0.2 eq.) were diluted with DMF (25 mL). After 2min, pyridine (5 mL) was added and the reaction was monitored usingreverse-phase HPLC. The reaction was shown to be complete in 24 h. Thereaction mixture was concentrated to provide a dark oil, which wasdiluted with 3 mL of DMF. The DMF solution was purified using flashcolumn chromatography (silica gel, eluant gradient: 100% dichloromethaneto 4:1 dichloromethane-MeOH). The relevant fractions were combined andconcentrated to provide an oil that solidified under high vacuum toprovide a mixture of the desired drug-linker compound and unreacted MMAEas a dirty yellow solid (R_(f) 0.40 in 9:1 dichloromethane-MeOH). Thedirty yellow solid was diluted with DMF and purified using reverse-phasepreparative-HPLC (Varian Dynamax C₁₈ column 41.4 mm×25 cm, 8μ, 100 Å,using a gradient run of MeCN and 0.1% aqueous TFA at 45 mL/min from 10%to 100% over 40 min followed by 100% MeCN for 20 min) to provide thedesired drug-linker compound as an amorphous white powder (Rf 0.40 in9:1 dichloromethane-MeOH) which was >95% pure by HPLC and whichcontained less than 1% of MMAE. Yield: 1.78 g (57%); ES-MS m/z 1316.7[M+H]⁺; UV λ_(max) 215, 248 nm.

10.1.2 Conjugate Preparation

Antibody Reduction. To 4.8 mL cAC10 (10 mg/mL) was added 600 μL of 500mM sodium borate/500 mM NaCl, pH 8.0, followed by 600 μL of 100 mM DTTin water. After incubation at 37° C. for 30 min, the buffer wasexchanged by elution over G25 resin equilibrated and eluted with PBScontaining 1 mM DTPA (Aldrich). The thiol/Ab value was checked bydetermining the reduced antibody concentration from the solution's 280nm absorbance, and the thiol concentration by reaction with DTNB(Aldrich) and determination of the absorbance at 412 nm.

Conjugation of the Reduced Antibody. The reduced mAb was chilled on ice.The drug-linker compound was used as a DMSO solution of knownconcentration, and the quantity of drug-linker added to the reactionmixture was calculated as follows: L stock solution=V×[Ab]×FoldExcess/[Drug-Linker], where V and [Ab] are the volume and molarconcentration of the reduced antibody solution, respectively. 2.3 mLcold PBS/DTPA was added to the reduced antibody solution. 133.6 uL of7.5 mM drug-linker compound stock solution was diluted into 1.47 mLacetonitrile. The acetonitrile drug-linker solution was chilled on ice,then added to the reduced antibody solution. The reaction was terminatedafter 1 hr by the addition of a 20 fold molar excess of cysteine overmaleimide. The reaction mixture was concentrated by centrifugalultrafiltration and purified by elution through de-salting G25 in PBS.cAC10-vcMMAE was then filtered through 0.2 micron filters under sterileconditions and immediately frozen at −80C. cAC10-vMMAE was analyzedfor 1) concentration, by UV absorbance; 2) aggregation, by sizeexclusion chromatography; 3) drug/Ab, by measuring unreacted thiols withDTNB, and 4) residual free drug, by reverse phase HPLC.

10.2 Synthesis of cAC10-fkAEFP

The synthesis of the ADC cAC10-fkAEFP, the general structure of which isdepicted above, is described below.

10.2.1 AEFP Synthesis

Boc-phenylalanine (1.0 g, 3.8 mmol) was added to a suspension of1,4-diaminobenzene.HCl (3.5 g, 19.0 mmol, 5.0 eq.) in triethylamine(10.7 mL, 76.0 mmol, 20 eq.) and dichloromethane (50 mL). To theresulting solution was added DEPC (3.2 mL, 19.0 mmol, 5.0 eq.) viasyringe. HPLC showed no remaining Boc-phe after 24 h. The reactionmixture was filtered, and the filtrate was concentrated to provide adark solid. The dark solid residue was partitioned between 1:1EtOAc-water, and the EtOAc layer was washed sequentially with water andbrine. The EtOAc layer was dried and concentrated to provide a darkbrown/red residue that was purified using HPLC (Varian Dynamax column41.4 mm×25 cm, 5μ, 100 Å, using a gradient run of MeCN and water at 45mL/min form 10% to 100% over 40 min followed by 100% MeCN for 20 min).The relevant fractions were combined and concentrated to provide ared-tan solid intermediate. Yield: 1.4 g (100%); ES-MS mil: 355.9[M+H]⁺; UV λ_(max) 215, 265 nm; ¹H NMR (CDCl₃) δ 7.48 (1H, br s),7.22-7.37 (5H, m), 7.12 (2H, d, J=8.7 Hz), 7.61 (2H, d, J=8.7 Hz), 5.19(1H, br s), 4.39-4.48 (1H, m), 3.49 (2H, s), 3.13 (2H, d, J=5.7 Hz),1.43 (9H, s).

The red-tan solid intermediate (0.5 g, 1.41 mmol) anddiisopropylethylamine (0.37 mL, 2.11 mmol, 1.5 eq.) were diluted withdichloromethane (10 mL), and to the resulting solution was added Fmoc-Cl(0.38 g, 1.41 mmol). The reaction was allowed to stir, and a white solidprecipitate formed after a few minutes. Reaction was complete accordingto HPLC after 1 h. The reaction mixture was filtered, and the filtratewas concentrated to provide an oil. The oil was precipitated with EtOAc,resulting in a reddish-white intermediate product which was collected byfiltration and dried under vacuum. Yield: 0.75 g (93%); ES-MS m/z 578.1[M+H]⁺, 595.6 [M+NH₄]⁺.

The reddish-white intermediate (0.49 g, 0.85 mmol), was diluted with 10mL of dichloromethane, and then treated with 5 mL of trifluoroaceticacid. Reaction was complete in 30 nin according to reverse-phase HPLC.The reaction mixture was concentrated and the resulting residue wasprecipitated with ether to provide an off-white solid. The off-whitesolid was filtered and dried to provide an amorphous powder, which wasadded to a solution of Boc-Dolaproine (prepared as described inTetrahedron, 1993, 49(9):1913-1924) (0.24 g, 0.85 mmol) indichloromethane (10 mL). To this solution was added triethylamine (0.36mL, 2.5 mmol, 3.0 eq.) and PyBrop (0.59 g, 1.3 mmol, 1.5 eq.). Thereaction mixture was monitored using reverse-phase HPLC. Uponcompletion, the reaction mixture was concentrated, and the resultingresidue was diluted with EtOAc, and sequentially washed with 10% aqueouscitric acid, water, saturated aqueous sodium bicarbonate, water andbrine. The EtOAc layer was dried (MgSO₄), filtered, and concentrated.The resulting residue was purified using flash column chromatography(silica gel) to provide an off-white powdered intermediate. Yield: 0.57g (88%); ES-MS m/z 764.7 [M+NH₄]⁺; UV λ_(max) 215, 265 nm; ¹H NMR(DMSO-d₃) δ 10.0-10.15 (1H, m), 9.63 (1H, br s), 8.42 (½H, d, J=8.4 Hz),8.22 (½H, d J=8.4 Hz), 7.89 (2H, d, J=7.2 Hz), 7.73 (2H, d, J=7.6,Hz),7.11-7.55 (13H, m), 4.69-4.75 (1H, m), 4.46 (2H, d, J=6.8 Hz), 4.29 (1H,t, J=6.4 Hz), 3.29 (3H, s), 2.77-3.47 (7H, m), 2.48-2.50 (3H, m), 2.25(⅔H, dd, J=9.6, 7.2 Hz), 1.41-1.96 (4H, m), 1.36 (9H, s), 1.07 (1H, d,J=6.4 Hz, rotational isomer), 1.00 (1H, d, J=6.4 Hz, rotational isomer).

The white solid intermediate (85 mg, 0.11 mmol) andMe-val-val-dil-O-t-butyl (55 mg, 0.11 mmol, prepared as described inPettit et al. J. Chem. Soc. Perk. 1, 1996, 859) were diluted withdichloromethane (5 mL), and then treated with 2.5 mL of trifluoroaceticacid under a nitrogen atmosphere for two hours at room temperature. Thereaction completion was confirmed by RP-HPLC. The solvent was removed invacuo and the resulting residue was azeotropically dried twice withtoluene, then dried under high vacuum for 12 hours.

The residue was diluted with dichloromethane (2 mL),diisopropylethylamine (3 eq.) was added, followed by DEPC (1.2 eq.).After the reaction was completed, the reaction mixture was concentratedunder reduced pressure, the resulting residue was diluted with EtOAc,and washed sequentially with 10% aqueous citric acid, water, saturatedaqueous sodium bicarbonate, and brine. The EtOAc layer was dried,filtered and concentrated to provide a yellow oil.

The yellow oil was diluted with dichloromethane (10 mL) and to theresulting solution diethylamine (5 mL) was added.” According to HPLC,reaction was completed after 2 h. The reaction mixture was concentratedto provide an oil. The oil was diluted with DMSO, and the DMSO solutionwas purified using reverse phase preparative-HPLC (Varian Dynamax column21.4 mm×25 cm, 5μ, 100 Å, using a gradient run of MeCN and 0.1% TFA at20 mL/min from 10% to 100% over 40 min followed by 100% MeCN for 20min). The relevant fractions were combined and concentrated to providethe desired drug as an off-white solid. Overall yield: 42 mg (44%overall); ES-MS m/z 837.8 [M+H]⁺, 858.5 [M+Na]⁺; UV λ_(max) 215, 248 nm.

10.2.2 Linker Synthesis

The linker compound maleimidocaproyl-L-phenylalanine-L-lysine(MMT) wasprepared as described in Dubowchik et al., 2002, Bioconjugate Chem.13:855-896.

10.2.3 Preparation of Drug-Linker Compound

The drug of Section 10.2.1 (9 mg, 10.8 mmol) and the linker from section10.2.2 (5.2 mg, 10.8 μmol) were diluted with dichloromethane (1 mL) andto the resulting solution was added HATU (6.3 mg, 16.1 mmol, 1.5 eq.),followed by pyridine (1.3 μL, 16.1 μmol, 1.5 eq.). The reaction mixturewas allowed to stir under argon atmosphere while being monitored usingHPLC. The reaction was complete after 6 h. The reaction mixture wasconcentrated and the resulting residue was diluted with DMSO. The DMSOsolution was purified using reverse phase preparative HPLC (VarianDynamax column 21.4 m=×25 mm, 5 m, 100 Å, using a gradient run of MeCNand Et3N—CO2 (pH 7) at 20 mL/min from 10% to 100% over 40 min followedby 100% MeCN for 20 min) and the relevant fractions were combined andconcentrated to provide an off-white solid intermediate which was >95%pure according to HPLC.

The off-white solid intermediate was diluted with dichloromethane (2 mL)and the resulting solution was treated with TFA (0.5 mL). The reactionwas monitored using HPLC, and was complete in 2 h. The reaction mixturewas concentrated, and the resulting residue was diluted with DMSO andpurified under the same conditions as described in Example 13. Therelevant fractions were combined and concentrated to provide the desireddrug-linker compound as an off-white powder. Yield: 14.9 mg (90%); ES-MSm/z 1304.6 [M+H]⁺; UV λ_(max) 215, 275 nm.

10.2.4 Conjugate Preparation

Antibody Reduction. To 3.0 mL cAC10 (10 mg/mL) was added 375 μL of 500mM sodium borate/500 mM NaCl, pH 8.0, followed by 375 μL of 100 mM DTTin water. After incubation at 37° C. for 30 min., the buffer wasexchanged by elution over G25 resin equilibrated and eluted with PBScontaining 1 mM DTPA (Aldrich). The thiol/Ab value was checked bydetermining the reduced antibody concentration from the solution's 280nm absorbance, and the thiol concentration by reaction with DTINB(Aldrich) and determination of the absorbance at 412 nm.

Conjugation of the Reduced Antibody. The reduced mAb was chilled on ice.The drug-linker compound was used as a DMSO solution of knownconcentration, and the quantity of drug-linker added to the reactionmixture was calculated as follows: L stock solution=V×[Ab]×FoldExcess/[Drug-Linker], where V and [Ab] are the volume and molarconcentration of the reduced antibody solution, respectively. 2.2 mLcold PBS/DTPA was added to the reduced antibody solution, followed by1.47 mL DMSO, and the mixture chilled on ice. 140.0 uL of 7.6 mMdrug-linker compound stock solution was then added to the reducedantibody/ DMSO solution. The reaction was terminated after 1 hr by theaddition of a 20 fold molar excess of cysteine over maleimide. Thereaction mixture was concentrated by centrifugal ultrafiltration andpurified by elution through de-salting G25 in PBS. cAC10-fkAEFP was thenfiltered through 0.2 micron filters under sterile conditions andimmediately frozen at −80° C. cAC10-fkAEFP was analyzed for 1)concentration, by UV absorbance; 2) aggregation, by size exclusionchromatography; 3) drug/Ab, by measuring unreacted thiols by treatmentwith DTT, followed by DTNB, and 4) residual free drug, by reverse phaseHPLC.

10.3 Synthesis of cAC10-vcAEFP:

The synthesis of the ADC cAC10-vcAEFP, the general structure of which isdepicted above, is described below.

10.3.1 Drug Synthesis

The drug was synthesized as illustrated above herein at Section 10.2.1.

10.3.2 Preparation of Drug-Linker Compound

The trifluoroacetate salt of the drug of Section 10.2.1 (0.37 g, 0.39mmol, 1.0 eq.) and Fmoc-val-cit (0.30 g, 0.58 mmol, 1.5 eq., preparedaccording to Dubowchik et al., Bioconjugate Chem. 2002, 13, 855-896)were diluted with DMF (5 mL, 0.1 M), and to the resulting solution wasadded pyridine (95 μL, 1.2 mmol, 3.0 eq.). HATU (0.23 g, 0.58 mmol, 1.5eq.) was then added as a solid and the reaction mixture was allowed tostir under argon atmosphere while being monitored using HPLC. Thereaction progressed slowly, and 4 h later, 1.0 eq. ofdiisopropylethylamine was added. Reaction was complete in 1 h. Thereaction mixture was concentrated in vacuo and the resulting residue waspurified using prep-HPLC (Varian Dynamax C18 column 41.4 mm×25 cm, 5μ,100 Å, using a gradient run of MeCN and 0.1% aqueous TFA at 45 mL/minfrom 10% to 100% over 40 min followed by 100% MeCN for 20 min) toprovide a faint pink solid intermediate.

The pink solid intermediate was diluted with DMF (30 mL) and to theresulting solution was added diethylamine (15 mL). Reaction was completeby HPLC in 2 h. The reaction mixture was concentrated and the resultingresidue was washed twice with ether. The solid intermediate was driedunder high vacuum and then used directly in the next step.

The solid intermediate was diluted with DMF (20 mL) and to the resultingsolution was added 6-(2,5-dioxy-2,5-dihydro-pyrrol-1-yl)-hexanoic acid2.5-dioxy-pyrrolidin-1-yl ester (0.12 g, 0.39 mmol, 1.0 eq.) (EMCS,Molecular Biosciences Inc., Boulder, Colo.). After 4 d, the reactionmixture was concentrated to provide an oil which was purified usingprep-HPLC (Varian Dynamax C18 column 41.4 mm×25 cm, 5μ, 100 Å, using agradient run of MeCN and 0.1% aqueous TFA at 45 mL/min from 10% to 100%over 40 min followed by 100% MeCN for 20 min) to provide the desireddrug-linker compound as a white flaky solid. Yield: 0.21 g (38%overall); ES-MS m/z 1285.9 [M+H]+; 13.07.8 [M+Na]+; UV λ_(max) 215, 266nm.

10.3.3 Conjugate Preparation

Antibody Reduction. To 3.0 mL cAC10 (10 mg/mL) was added 375 μL of 500mM sodium borate/500 mM NaCl, pH 8.0, followed by 375 μL of 100 mM DTTin water. After incubation at 37° C. for 30 min., the buffer wasexchanged by elution over G25 resin equilibrated and eluted with PBScontaining 1mM DTPA (Aldrich). The thiol/Ab value was checked bydetermining the reduced antibody concentration from the solution's 280nm absorbance, and the thiol concentration by reaction withDTNB(Aldrich) and determination of the absorbance at 412 nm.

Conjugation of the Reduced Antibody. The reduced mAb was chilled on ice.The drug-linker compound was used as a DMSO solution of knownconcentration, and the quantity of drug-linker added to the reactionmixture was calculated as follows: L stock solution=V×[Ab]×FoldExcess/[Drug-Linker], where V and [Ab] are the volume and molarconcentration of the reduced antibody solution, respectively. 2.2 mLcold PBS/DTPA was added to the reduced antibody solution., followed by1.47 mL DMSO, and the mixture chilled on ice. 140.0 μL of 7.6 mMdrug-linker compound stock solution was then added to the reducedantibody/DMSO solution. The reaction was terminated after 1 hr by theaddition of a 20 fold molar excess of cysteine over maleimide. Thereaction mixture was concentrated by centrifugal ultrafiltration andpurified by elution through de-salting G25 in PBS. cAC10-vcAEFP was thenfiltered through 0.2 micron filters wider sterile conditions andimmediately frozen at −80° C. cAC10-vcAEFP was analyzed for 1)concentration, by UV absorbance; 2) aggregation, by size exclusionchromatography; 3) drug/Ab, by measuring unreacted thiols by treatmentwith DTT, followed by DTNB, and 4) residual free drug, by reverse phaseHPLC.

10.4 Synthesis of cAC10-fkMMAE

The synthesis of the ADC cAC10-fkMMAE, the general structure of which isdepicted above, is described below.

10.4.1 Preparation of Drug-Linker Compound

The synthesis of auristatin E has been previously described (Pettit G R,and Barkoczy, J., 1997 U.S. Pat. No. 5,635,483, Pettit, G. R., Prog.Chem. Org. Nat. Prod., 70, 1-79, 199). The monomethyl derivative ofAuristatin E (MMAE) was prepared by replacing a protected form ofmonomethylvaline for N,N-dimethylvaline in the synthesis of auristatin E(Senter et al., U.S. provisional application No. 60/400,403 filed Jul.31, 2002, which is incorporated by reference herein in its entirety).

MMAE (100 mg, 0.14 mmol), the linkermaleimidocaproyl-L-phenylalanine-L-lysine(MMT)-p-aminobenzyl alcoholp-nitrophenylcarbonate (160 mg, 0.15 mmol, 1.1 eq., prepared asdescribed in Dubowchik et al., Bioconjugate Chem. 2002, 13, 855-869),and HOBt (19 mg, 0.14 mmol, 1.0 eq.) were diluted with DMF (2 mL). After2 min, pyridine (0.5 mL) was added and the reaction mixture wasmonitored using reverse-phase HPLC. Neither MMAE nor the linker wasdetected after 24 h. The reaction mixture was concentrated, and theresulting residue was purified using reverse phase preparative-HPLC(Varian Dynamax column 21.4 mm×25 cm, 5μ, 100 Å, using a gradient run ofMeCN and Et₃N—CO₂ (pH 7) at 20 mL/min from 10% to 100% over 40 minfollowed by 100% MeCN for 20 min). The relevant fractions were pooledand concentrated to provide an off-white solid intermediate. ES-MS m/z1608.7 [M+H]⁺.

The off-white solid intermediate was diluted with MeCN/water/TFA in an85:5:10 ratio, respectively. The reaction mixture was monitored usingHPLC and was complete in 3 h. The reaction mixture was directlyconcentrated and the resulting residue was purified using reverse phasepreparative-HPLC (Varian Dynamax column 21.4 mm×25 cm, 5μ, 100 Å, usinga gradient run of MeCN and 0.1% TFA at 20 mL/min from 10% to 100% over40 min followed by 100% MeCN for 20 min). The relevant fractions werecombined and concentrated to provide the desired drug-linker compound asan off-white powder. Yield: 46 mg (32% overall); ES-MS m/z 1334.8[M+H]⁺; UV λ_(max) 215, 256 nm.

10.4.2 Conjugate Preparation

Antibody Reduction. To 4.8 mL cAC10 (10 mg/mL) was added 600 uL of 500mM sodium borate/500 mM NaCl, pH 8.0, followed by 600 uL of 100 mM DTTin water. After incubation at 37° C. for 30 min, the buffer wasexchanged by elution over G25 resin equilibrated and eluted with PBScontaining 1 nM DTPA (Aldrich). The thiol/Ab value was checked bydetermining the reduced antibody concentration from the solution's 280nm absorbance, and the thiol concentration by reaction with DTNB(Aldrich) and determination of the absorbance at 412 nm.

Conjugation of the Reduced Antibody. The reduced mAb was chilled on ice.The drug-linker compound was used as a DMSO solution of knownconcentration, and the quantity of drug-linker added to the reactionmixture was calculated as follows: L stock solution=V×[Ab]×FoldExcess/[Drug-Linker], where V and [Ab] are the volume and molarconcentration of the reduced antibody solution, respectively. 2.3 mLcold PBS/DTPA was added to the reduced antibody solution. 133.6 uL of7.5 drug-linker compound stock solution was diluted into 1.47 mLacetonitrile. The acetonitrile drug-linker solution was chilled on ice,then added to the reduced antibody solution. The reaction was terminatedafter 1 hr by the addition of a 20 fold molar excess of cysteine overmaleimide. The reaction mixture was concentrated by centrifugalultrafiltration and purified by elution through de-salting G25 in PBS.cAC10-fkMMAE was then filtered through 0.2 micron filters under sterileconditions and immediately frozen at −80C. cAC10-fkMMAE was analyzedfor 1) concentration, by UV absorbance; 2) aggregation, by sizeexclusion chromatography; 3) drug/Ab, by measuring unreacted thiols withDTNB, and 4) residual free drug, by reverse phase HPLC.

10.5 Results

The ability of cAC10-vcMMAE to inhibit Jurkat T cell proliferation wasthen examined. Five thousand Jurkat T cells in 100 μl of medium wereseeded in 96-well TC plates. Graded concentrations of cAC10-vcMMAE or acontrol non-binding control IgG (cIgG)-vcMMAE in 100 μl of medium wereadded to Jurkat cells to achieve the final concentrations given in FIG.9. After 80 hours of incubation, cellular DNA synthesis was assessed bya 16 hour ³H-TdR pulse. Chimeric AC10-vcMMAE at concentrations higherthan 0.001 μg/ml significantly inhibited DNA synthesis. The IC₅₀ wasapproximately 0.1 μg/ml. The control cIgG-vcMMAE did not showsignificant growth inhibitory action at concentrations lower than 0.1μg/ml. FIG. 2 shows that soluble, unconjugated cAC10 had growthinhibitory effect on Jurkat cells at concentration higher than 1 μg/ml.Collectively, these data suggest that cAC10 is effective in delivering acytotoxic drug to CD30⁺ target cells, and that the cAC10-vcMMAE ADCefficiently internalized upon binding to target cells.

11. EXAMPLE 6 Activation of Normal Human Peripheral Blood MononuclearCells (PBMC) Induced CD30 Expression on T Lymphocytes

Human PBMC obtained from normal donors through apheresis were the sourceof human T lymphocytes. Immobilized anti-CD3 and anti-CD28 mAbs wereused to activate T lymphocytes and induce them to proliferate. Toimmobilize mAbs, one μg/ml of each an anti-CD3 mAb (OKT3) and ananti-CD28 mAb (B-T3, DiaClone Research, Besancon, France, or 9.3, Haraet al., 1985, J. Exp. Med., 161, 1513-1524) in PBS were incubated at 37C for 2 hours in TC wells or flasks. Unbound mAbs were removed by twowashes with PBS. T cell activation was initiated by seeding PBMC intomAb-coated TC wells or flasks at a concentration of 0.5×10⁶ cells/ml inculture medium (RPMI-1640, 10% FBS, 2 mM L-glutamine, 1 mM sodiumpyruvate, and 0.1 mM non-essential amino acids). In some experiments,recombinant human IL-2 (rhIL-2) (Chiron, Emeryville, Calif.) at a finalconcentration of 100 IU/ml was also included.

T cell activation and CD30 expression were monitored by multi-color flowcytometric analysis. For detecting CD30 expression, purified anti-CD30(AC10) was labeled with the fluorescent probe Alexa Fluor (AF)(Molecular Probes, Eugene, Oreg.) according to the manufacturer'sinstruction. Before antibody staining, activated PBMC were washed oncein staining medium (PBS, 1% bovine serum albumin (BSA), 0.02% sodiumazide) and pelleted. A 25 μl cocktail of either AF labeled anti-CD3+phycoerythrin (PE) labeled anti-CD4 (BD PharMingen, San Diego,Calif.)+Cy-Chrome (Cy) labeled anti-CD8 (BD PharMingen) or fluoresceinisothiocyanate (FITC) anti-CD25 (BD PharMingen)+PE labeled anti-CD4 (BDPharMingen)+Cy labeled anti-CD8 (BD PharMingen) in staining medium wasused to resuspend a 0.2×10⁶ cell pellet. Cells were incubated on ice for20 minutes, washed 2 times w ith staining medium, and fixed in 1%paraformaldehyde in PBS before being analyzed on a FACscan (BDBioscience, San Jose, Calif.).

As shown in FIG. 10, very few T lymphocytes in unstimulated PBMCexpressed CD30. Activation of PBMC with either anti-CD3+anti-CD28 oranti-CD3+anti-CD28′ IL-2 induced a time-dependent expression of CD30 onT lymphocytes. In contrast, PBMC maintained in medium alone did not showinduction of CD30 expression. Increased expression of CD30 was detectedon both CD4⁺ and CD4⁻ (containing CD8⁺ cells, data not shown) cells. Thepeak of expression on both CD4⁺ and CD8⁺ T lymphocytes was around 4 daysafter stimulation. The level declined thereafter to almost basal after 8days in culture.

12. EXAMPLE 7 cAC10 ADCS Inhibited Proliferation of Activated Normal TLymphocytes

The ability of cAC10 ADCs to inhibit the proliferation of activatednormal T lymphocytes was then examined. Normal PBMCs were activated byanti-CD3 and anti-CD28 mAbs as described in Example 6. On day 3 duringculture, which was close to the peak of CD30 induction (FIG. 10),activated PBMC were harvested. A portion of the cells was used for flowcytometric analysis to confirm the CD30 expression on both CD4 and CDScells. The rest of the cells were pelleted and resuspended in freshmedium containing 200 IU/ml of rhIL-2 at 50,000 cells/ml. One hundred μlof this cell suspension (5,000 cells) were transferred to each well of96-well TC plates. One hundred μl of graded concentrations ofcAC10-fkMMAE, cAC10-vcMMAE, or the corresponding non-binding cIgG ADCsin medium supplemented with 200 IU/ml of rhIL-2 were added to culturewells to achieve the concentrations indicated in FIG. 11. Cells werecultured for an additional 48 or 72 hours with a pulse of ³H-TdR duringthe last 16 hours to assess cellular DNA synthesis. Similar to itsactivity on Jurkat T cells, cAC10 ADCs inhibited the proliferation ofactivated PBMC (FIG. 11). Inhibitory effects were detectable when cAC10ADCs were present at concentrations higher than 0.01 μg/ml. A 72-hourincubation with either ADC resulted in more profound growth inhibitionthan a 48-hour incubation. Chimeric AC10-fkMMAE appeared to be moreactive than cAC10-vcMMAE.

13. EXAMPLE 8 Memory T Lymphocytes were More Sensitive to cAC10 ADC thanNaïve T Lymphocyte

As PBMC are made up of different subsets of lymphocytes, the response ofone cellular subset toward cAC10 ADCs may easily be masked by othersubsets. In order to examine this possibility, total T lymphocytes,naïve and memory T lymphocytes were enriched from PBMC by negativeimmuno-selection. Briefly, PBMCs were incubated with one of thefollowing antibody cocktails, containing saturating quantities ofantibodies, at a final concentration of 20×10⁶ cells/ml on ice for 20minutes. To enrich for total T lymphocytes, the antibody cocktailcontained anti-CD 14 (BD PharMingen), anti-CD16 (BD PharMingen), andanti-CD20 (BD PharMingen). To enrich for naïve T lymphocytes (CD45RO⁻),the antibody cocktail contained anti-CD14, anti-CD16, anti-CD20, andanti-CD45RO (BD PharMingen). To enrich for memory T lymphocytes(CD45RA⁻), the antibody cocktail contained, anti-CD14, anti-CD16,anti-CD20, and anti-CD45RA (BD PharMingen). All antibody cocktails wereprepared in medium supplemented with 10% FBS. After antibody binding,cells were washed twice with ice-cold culture medium and resuspended toa concentration of 20×10⁶ cells/ml in culture medium. Antibody-boundcells were removed from the cell suspension by Dynabeads M450 goatanti-mouse IgG paramagnetic beads (Dynal, Oslo. Norway). Before additionto the cell suspension, Dynabeads M450 goat anti-mouse IgG were washedtwice by culture medium and resuspended to a concentration of 60×10⁶/mlin culture medium. Equal volumes of cell and paramagnetic beads weremixed and rotated for 2 hours at 4° C. The cell/paramagnetic beadsuspension was diluted 2-fold with culture medium. Unbound paramagneticbeads and paramagnetic bead-cell conjugates were attracted to the sideof the culture by the application of a magnet. Unbound cells, enrichedfor the subsets described above were removed. Flow cytometric analysiswas conducted to confirm the enrichment as shown on the left column ofFIG. 12.

Activation-induced expression of CD30 on different T cell subsets wasthen examined. Briefly, total, naïve, and memory T lymphocytes obtainedfrom immuno-selection were stimulated by immobilized anti-CD3 andanti-CD28 antibodies in the presence of 200 IU/ml of IL-2 as describedin Example 6. After 72 hours of incubation, the expression of CD30 onboth CD4 and CDS cells in the different cultures was determined by flowcytometry, also as outlined in Example 6. Expression of CD30 wasdetected on both CD4 and CDS cells regardless of whether the startingpopulation was total, naïve, or memory T lymphocytes (FIG. 12). Thelevels of CD30 on memory CD4⁺ and CD8⁺ cells were slightly higher thanthe other subsets on a consistent basis.

The responses of activated naïve (CD45RO⁻) and memory T lymphocytes(CD45RA⁻) to cAC10 ADCs were then examined. Three-day activated naïveand memory T lymphocytes were plated at 5,000 cells/well in 96-well TCplates in a final volume of 200 μl of medium containing 200 IU/ml ofrhIL-2 and cAC10 ADCs or cIgG ADCs at final concentrations indicated inFIG. 13. Cells were incubated for an additional 72 hours with the last16 hours pulsed with ³H-TdR to assess cellular DNA synthesis. WhereascIgG ADCs did not have any significant effect on T cell proliferation atconcentrations lower than 2 μg/ml, proliferation of activated memory Tlymphocytes was significantly inhibited by either cAC10-vcMMAE orcAC10-vcAEFP at concentrations higher than 0.007 μg/ml. By contrast,activated naïve T lymphocytes were relatively refractory tocAC10-vcMMAE. The cAC10-vcAEFP conjugate was found to be more effectivethan the cAC10-vcMMAE conjugate on both T cell subsets. Notwithstanding,naïve T lymphocytes were still less sensitive to cAC10-vcAEFP thanmemory T lymphocytes (FIG. 13). The difference in sensitivity betweennaïve and memory T lymphocytes toward the cAC10 ADCs was probably not aconsequence of antigen densities, as both activated naïve and memory Tlymphocytes were found to express comparable levels of CD30 (FIG. 12).The growth inhibitory activity of cAC10-vcMMAE reported in FIG. 11probably reflected the combined responses of naïve and memory Tlymphocytes. More importantly, these results suggest that cAC10-vcMMAEmay be able to selectively suppress CD30⁺ memory T lymphocyteproliferation while having minimal effects on CD30⁻ naïve T lymphocytes.Effector T lymphocytes implicated in the pathogenesis of autoimmune,inflamatory, and allergic diseases are usually antigen-primed, and theybelong to the memory T lymphocyte subset. Accordingly, application ofcAC10-vcMMAE in therapeutic intervention may have the advantage of onlytargeting antigen-primed T lymphocytes. On the other hand, applicationof cAC10-vcAEFP may be preferred in situations in which suppressing theproliferation of both activated naïve and memory T lymphocytes isdesired, e.g., during transplant rejection.

14. EXAMPLE 9 Allogeneic Stimulation of Normal Human T LymphocytesInduced CD30 Expression

Effector T lymphcytes implicated in the pathogenesis of autoimmune,allergic, and inflamatory responses have usually gone through multiplerounds of antigenic stimulation and expansion. During this process ofchronic activation, they continue to carry out effector functionsincluding cytokine secretion and cytolytic responses to induce tissuedamages. It is therefore important to examine if T lymphocytes that haveundergone repeated rounds of activation and expansion can still expressCD30, and how anti-CD30 mAbs and their ADCs can be applied to inhibitthe expansion of such chronically activated CD30⁺ T lymphocytes. One ofsuch T cell lines was generated against allogeneic stimulator cells.CD4⁺ lymphocytes were enriched from PBMC by the depletion of CD8⁺ cellsas detailed in Example 9. Briefly, PBMC were incubated in culture mediumcontaining a saturating concentration of an anti-CD8 mAb (BD PharMingen)on ice for 20 minutes. Cells were then washed twice with ice-coldculture medium. Anti-CD8 bound cells were removed from the cellsuspension by Dynabeads M450 goat anti-mouse IgG paramagnetic beads.Unbound cells, enriched for CD4′ cells, were analyzed by flow cytometryfor CD4 expression.

PBMC (5×10⁶), enriched for CD4⁺ cells, were co-cultured with an equalnumber of gamma-irradiated (2700-5000 rads) allogeneic Daudi cells(ATCC). EL-2 was added to a final concentration of 400 IU/ml on day 4,and the culture was allowed to continue until day 14. Viable cells werethen re-stimulated and expanded with irradiated Daudi cells at a T cellto Daudi ratio of 1:3 in the presence of 200 IU/ml of IL-2. This was thebeginning of cycle 2 (FIG. 14). After 7 to 9 days in culture, Tlymphocytes were re-stimulated again with irradiated Daudi cell to startthe following round of expansion. Expression of CD30 was examined 3 to 4days and 7 to 9 days after the addition of allogeneic stimulator cells.As shown in FIG. 7, CD30 was induced in each round of allogeneicstimulation on both the CD4 and CD4- cells. The levels of CD30 expressedgradually decline toward the end of each stimulation cycle. Theseresults confirm that antigen-primed T lymphocytes retain the capacity toexpress CD30 when they are challenged with antigenic stimulation.

15. EXAMPLE 10 Generation of T Lymphocyte Clones

An alternative way to simulate effector lymphocytes in vitro is togenerate T lymphocyte clones. FIG. 16 depicts a protocol for generatingantigen non-specific helper (Th) and cytotoxic (Tc) T lymphocytesclones. PBMC were used as the starting material. CD4⁺ or CD8⁺ cells wereenriched using the immuno-selection methods described in Examples 8 and9. Two approaches were applied to stimulate and expand T lymphocytes. Inthe first approach, cells were stimulated with immobilized anti-CD3 plussoluble anti-CD28 and rhIL-2 (200 IU/ml) at limiting dilution in 96-wellTC plates. Anti-CD3 was immobilized onto TC wells at one μg/ml asdescribed in Example 6. Alternatively, cells were stimulated withphytohemagglutinin (Sigma, 1-2 μg/ml), 10,000 irradiated CESS cells(ATCC, 2700 rad irradiated), and rhIL-2 (200 IU/ml) at limiting dilutionin 96-well plates. Additional supplements, including differentcombinations of cytokines and/or antibodies against cytokines can beused to skew the development of T lymphocyte clones to Th₁ or Th₂effector cells.

Clones identified from the limiting dilution assays were furtherexpanded by multiple rounds of re-stimulation; each round lasted for 10to 12 days. During the second round of expansion, clones were stimulatedwith PHA (1-2 μg/ml), 1×10⁶ irradiated feeder cells (CESS), and rhIL-2(200 IU/ml). In some experiments, anti-IL-12 (R&D Systems, 5 μg/ml) andLX (R&D Systems, 10 ng/ml) were supplemented to favor T lymphocyte clonedevelopment toward the Th₂ or Tc, subsets. During the subsequent roundsof re-stimulation a T lymphocytes:feeder cells ratio of 1:2 was used andIL-2 was supplemented at 200 IU/ml. Several panels of T cell clones weregenerated using these stimulation protocols, and they were subjected tofurther phenotypic and functional analysis.

16. EXAMPLE 11 Phenotype of T Lymphocyte Clones

The surface phenotypes of 10 T lymphocyte clones determined by flowcytometry were depicted in FIG. 16. All 10 clones expressed high levelsof CD3 and detectable levels of CD28. Six of the 10 clones expressedCD4, and they can be considered as T helper clones (Th). Three of theremaining four can be considered as cytotoxic T lymphocyte clones (Tc)as they expressed CD8. The last clone was CD4⁺/CD8⁺. Expression of CD30was detectable on all 10 clones. The magnitudes of signals wererelatively low, as the analysis was conducted on resting clones.

The T lymphocyte clones were also analyzed for cytokine expression (FIG.17). Resting clones were stimulated for 4 hours at 37° C. with leukocyteactivation cocktail (BD PharMingen). Golgi Plug (BD PharMingen) wasadded to enable accumulation of intracellular cytokine by blockingprotein secretion. Cells were then washed and fixed with theCytofix/Cytoperm Kit (BD PharMingen) according to the manufacturer'sinstruction. Intracellular cytokines were detected using variousanti-cytokine antibodies (BD PharMingen), and stained cells wereanalyzed by flow cytometric analysis. Two of the Th clones expressedIL-4, IL-13, and IFNγ; this is consistent with a Tk profile. Four Thclones showed Th₂ profiles, i.e., they expressed IL-4, IL-5, or IL-13,but not IFNγ. Two Tc clones showed Tc₀ cytokine profile and one Tc cloneshowed Tc₂ profile. The CD4⁺/CD8⁺ clone expressed IL-4, IL-5, and IL-13,but not IFNγ.

17. EXAMPLE 12 Re-Stimulation of T Lymphocyte Clones Induced ExpressionOF CD30

The expression of CD30 during re-activation of the T lymphocyte cloneswas examined. Resting T lymphocyte clones were stimulated with 1 or 2μg/ml of PHA, irradiated CESS feeder cells at a T:CESS cell ratio of 1:2to 1: 0, 200 IU/ml of IL-2. IL-4 at 20 ng/ml was also supplemented tothe Th₂ and Tc₂ clones. The expression of CD25 and CD30 was monitored byflow cytometric analysis. FIG. 18 shows the results from onerepresentative Th clone and one representative Tc clone. Extensiveupregulation of CD25 was observed in all clones that peaked on day 2.This is indicative of T lymphocyte activation. Expression of CD25gradually declined in the following days. The induction of CD30expression paralleled that of CD25. The peak induction was also observedafter 2 days of stimulation. Expression was still detectable on day 4and it gradually declined to almost basal level by day 7. The other 8clones examined also showed similar kinetics and magnitudes of CD25 andCD30 expression (data not shown). Activation-induced expression of bothCD25 and CD30 has been a consistent feature of these T lymphocyteclones.

18. EXAMPLE 13 cAC10 ADCS Inhibited the Proliferation of Activated TLymphocyte Clones

The responses of T lymphocyte clones to cAC10 ADCs were examined next.Resting T lymphocyte clones were activated to express CD30 as describedin Example 12. After 2 days of activation, a portion of the cells wasanalyzed for CD30 expression by flow cytometry to confirm cellularactivation and CD30 induction. The remaining cells were pelleted andresuspended in new medium containing 200 IU/ml of rIL-2 or 200 IU/ml ofIL-2 and 10 ng/ml IL-4 for the Th₂ and Tc₂ clones. Cells were thenplated out at 10,000 cells/well in a final volume of 200 μl of mediumcontaining graded concentrations of cAC10 ADCs or the non-binding IgGADCs as indicated in FIG. 19 and FIG. 20. Cells were incubated for anadditional 48 or 72 hours with the last 16 hours pulsed with ³H-TdR toassess cellular DNA synthesis. Results for the responses of two Th₂clones toward ADC treatment was shown in FIG. 19 and FIG. 20. ChimericAC10 ADCs at concentrations higher than 0.01 μg/ml significantlyinhibited the proliferation both clones. The control ADC cIgG-fkMMAE didnot significantly inhibit proliferation at concentrations below 0.1μg/ml, whereas for cIgG-vcMMAE concentrations as high as 2 μg/ml showedno growth inhibitory activity, confirming the antigen specificity of thecAC10 ADCs. An incubation of a total of 72 hours also resulted in muchprofound proliferation inhibition than a 48-hour incubation.

Annexin V binding and membrane permeability to PI described in Example 4were then used to assess if the inhibition of proliferation effected bythe cAC10 ADCs was accompanied by cell death (FIG. 21). After a 4S-hourincubation with 1 μg/ml of ADC, 27% of the cAC10-vcMMAE-treated cellswas either apoptotic or dead, compared to 7-14% of thecIgG-fkMMAE-treated cells. For the cAC10-fkMMAE-treated cells, 44-48%were apoptotic or dead, compared to 13 and 31% of the cIG-fkMMAE-treatedcells. These data confirmed that the cAC10 ADCs tested were cytotoxic toCD30⁺ T cell clones.

During the course of analyzing the effects of cAC10 ADCs on theproliferation of activated T lymphocyte clones, several clones werefound to be relatively refractory to cAC10-vcMMAE treatment, e.g.,clones 3.27.2 and 4.01.1 in FIG. 22. Similar to what was observed inmemory and naïve T cells (FIG. 13), an AEFP conjugate of cAC10 showedstrong inhibitory activity on the T lymphocyte clones. Thus, bothcAC10-fkAEFP and cAC10-vcAEFP conjugates gave IC₅₀'s of 0.01 to 0.1μg/ml, compared to >1 μg/ml from cAC10-vcMMAE. These data furtherconfirm the utility of different cAC10 ADCs in targeted inhibition ofproliferation in T lymphocytes.

19. EXAMPLE 14 Antigen-Primed T Lymphocytes were More Sensitive to cAC10ADCS

FIG. 23 summarizes the efficacies of cAC10-vcMMAE and cAC10-vcAEFP oninhibiting the proliferation of different types of T lymphocytes. Thefollowing trend was observed. First, naïve T cells appeared to be mostrefractory to both cAC10-vcMMAE and cAC10-vcAEFP when compared to memoryT lymphocytes or T lymphocyte clones. Second, between the T lymphocyteclones and memory T cells, eight of the 10 T lymphocyte clones werefound to be more sensitive to cAC10-vcMMAE compared to the memory Tlymphocytes. These clones have been expanded through multiple rounds ofT cell receptor and cytokine stimulation, similar to chronicallystimulated effector T cells involved in inflammatory and autoimmuneresponses. Third, the proliferation of both CD4⁺ and CD8⁺ T lymphocytesclones was susceptible to cAC10 ADCs. Fourth, susceptibility to cAC10ADC also did not appear to correlate to any particular T lymphocytesubsets as defined by their cytokine secretion profiles. Thus, Th₀, Th₂,and Tc₀ clones and one each of Tc₂ and CD4⁺/CD⁸⁺ clones were found to besensitive to cAC10 ADCs. These results suggest that cAC10 ADCs could beused to target multiple T lymphocyte subsets including naïve or memory Tlymphocytes, helper (CD4⁺) or cytotoxic (CD8⁺) lymphocytes, and effectorlymphocytes secreting different combinations of cytokines. Moreover,since effector T lymphocytes participating in the pathogenesis of immunedisorders are biologically more similar to the chronically activated Tlymphocyte clones than to naïve T cells, cAC10 ADCs may be particularlysuited for the targeted depletion of CD30⁻ effector T lymphocytesinvolved in the pathogenesis of autoimmune, inflammatory, and allergicresponses.

20. Specific Embodiments Citation of References

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

Various references, including patent applications, patents, andscientific publications, are cited herein, the disclosures of each ofwhich is incorporated herein by reference in its entirety.

1. A method for the treatment of an immunological disorder in a subject,wherein the immunological disorder is not cancer, comprisingadministering to the subject, in an amount effective for said treatment,a pharmaceutical composition comprising (a) a first antibody that (i)immunospecifically binds CD30 and (ii) exerts a cytostatic or cytotoxiceffect on an activated lymphocyte; and (b) a pharmaceutically acceptablecarrier.
 2. The method of claim 1, wherein the first antibody is human,humanized or chimeric.
 3. The method of claim 1, wherein the firstantibody is multivalent.
 4. The method of claim 1, wherein the firstantibody competes for binding to CD30 with monoclonal antibodies AC10 orHeFi-1.
 5. The method of claim 1, wherein the first antibody is capableof exerting the cytotoxic or cytostatic effect without conjugation to acytotoxic agent.
 6. The method of claim 1, wherein the first antibody iscapable of exerting the cytotoxic or cytostatic effect in the absence ofcells other than the activated lymphocyte.
 7. The method of claim 1,wherein the first antibody is capable of exerting the cytotoxic orcytostatic effect as a monospecific antibody.
 8. The method of claim 1,further comprising administering an agent that potentiates thecytostatic or cytotoxic effect of the first antibody.
 9. The method ofclaim 1, further comprising administering a second antibody.
 10. Themethod of claim 1, wherein the second antibody recognizes a secondreceptor or receptor complex expressed on activated lymphocytes.
 11. Themethod of claim 10, wherein the second antibody enhances the cytostaticor cytotoxic effect of the first antibody .
 12. The method of claim 11,wherein the second antibody enhances the cytostatic or cytotoxic effectof the first antibody by delivering a signal to the activatedlymphocyte.
 13. The method of claim 10 or 11, wherein the receptor orthe receptor complex comprises an immunoglobulin gene superfamilymember, a TNF receptor superfamily member, an integrin, a cytokinereceptor, a chemokine receptor, a major histocompatibility protein, alectin, or a complement control protein.
 14. The method of claim 13,wherein the immunoglobulin superfamily member is CD2, CD3, CD4, CD8,CD19, CD22, CD28, CD79, CD90, CD152/CTLA-4, PD-1, or ICOS.
 15. Themethod of claim 13, wherein the TNF receptor superfamily member is CD27,CD40, CD95/Fas, CD134/OX40, CD137/4-1BB, TNT-RL, TNFR-2, RANK, TACI,BCMA, osteoprotegerin, Apo2/TRAIL-R1, TRAIL-R-1, TRAIL-R3, TRAIL-R4, orAPO-3.
 16. The method of claim 13, wherein the integrin is CD11a, CD11b,CD 11c, CD18, CD29, CD41, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f,CD103, or CD104.
 17. The method of claim 13, wherein the lectin isC-type, S-type, or I-type lectin.
 18. The method of claim 1, wherein thefirst antibody is a bispecific antibody.
 19. The method of claim 18,wherein the wherein the bispecific antibody binds to CD30 and a secondreceptor or receptor complex expressed on activated lymphocytes.
 20. Themethod of claim 19, wherein the portion of the bispecific antibody thatbinds to the second receptor or receptor complex enhances the cytostaticor cytotoxic effect of the portion of the bispecific antibody that bindsto CD30.
 21. The method of claim 20, wherein the binding of thebispecific antibody to the second receptor or receptor complex enhancesthe cytostatic or cytotoxic effect of the of the portion of thebispecific antibody that binds to CD30 by delivering a signal to theactivated lymphocyte.
 22. The method of claim 19 or 20, wherein thereceptor or receptor complex comprises an immunoglobulin genesuperfamily member, a TNF receptor superfamily member, an integrin, acytokine receptor, a chemokine receptor, a major histocompatibilityprotein, a lectin, or a complement control protein.
 23. The method ofclaim 22, wherein the immunoglobulin superfamily member is CD2, CD3,CD4, CD8, CD19, CD22, CD28, CD79, CD90, CD152/CTLA-4, PD-1, or ICOS. 24.The method of claim 22, wherein the TNF receptor superfamily member isCD27, CD40, CD95/Fas, CD134/OX40, CD137/4-1BB, TNF-R1, TNFR-2, RANK,TACI, BCMA, osteoprotegerin, Apo2/TRAIL-R1, TRAIL-R2, TRAIL-R3,TRAIL-R4, or APO-3.
 25. The method of claim 22, wherein the integrin isCD11a, CD11b, CD11c, CD18, CD29, CD41, CD49a, CD49b, CD49c, CD49d,CD49e, CD49f, CD103, or CD104.
 26. The method of claim 22, wherein thelectin is C-type, S-type, or I-type lectin.
 27. The method of claim 1,further comprising administering a ligand that binds to a receptor orreceptor complex expressed on activated lymphocytes.
 28. The method ofclaim 27, wherein the ligand enhances the cytostatic or cytotoxic effectof the first antibody.
 29. The method of claim 28, wherein the ligandenhances the cytostatic or cytotoxic effect of the first antibody bydelivering a signal to the activated lymphocyte.
 30. The method of claim27 or 28, wherein the receptor or receptor complex comprises animmunoglobulin gene superfamily member, a TNF receptor superfamilymember, an integrin, a cytokine receptor, a chemokine receptor, a majorhistocompatibility protein, a lectin, or a complement control protein.31. The method of claim 30, wherein the immunoglobulin superfamilymember is CD2, CD3, CD4, CD8, CD19, CD22, CD28, CD79, CD90,CD152/CTLA-4, PD-1, or ICOS.
 32. The method of claim 30, wherein the TNFreceptor superfamily member is CD27, CD40, CD95/Fas, CD134/OX40,CD137/4-1BB, TNF-R1, TNFR-2, RANK, TACI, BCMA, osteoprotegerin,Apo2/TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4, or APO-3.
 33. The method ofclaim 30, wherein the integrin is CD11a, CD11b, CD11c, CD18, CD29, CD41,CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD103, or CD104.
 34. Themethod of claim 30, wherein the lectin is C-type, S-type, or I-typelectin.
 35. The method of claim 1, wherein the first antibody is afusion protein comprising the amino acid sequence of a second proteinthat is not an antibody.
 36. The method of claim 35, wherein the secondprotein confers multivalent binding properties to the first antibody.37. The method of claim 1, 9, 10, 18, or 27, further comprisingadministering an immunosuppressive agent.
 38. The method of claim 37,wherein the immunosuppressive agent is gancyclovir, etanercept,cyclosporine, tacrolimus, or rapamycin.
 39. The method of claim 37,wherein the immunosuppressive agent is an alkylating agent.
 40. Themethod of claim 39, wherein the alkylating agent is cyclophosphamide.41. The method of claim 37, wherein the immunosuppressive agent is anantimetabolite.
 42. The method of claim 41, wherein the antimetaboliteis a purine antagonist.
 43. The method of claim 42, wherein the purineantagonist is azathioprine, or mycophenolate mofetil.
 44. The method ofclaim 41, wherein the antimetabolite is a dihydrofolate reductaseinhibitor.
 45. The method of claim 44, wherein the dihydrofolatereductase inhibitor is methotrexate.
 46. The method of claim 37, whereinthe immunosuppressive agent is a glucocorticoid.
 47. The method of claim46, wherein the glucocorticoid is cortisol or aldosterone.
 48. Themethod of claim 37, wherein the immunosuppressive agent is aglucocorticoid analogue.
 49. The method of claim 48, wherein theglucocorticoid analogue is prednisone or dexamethasone.
 50. The methodof claim 37, wherein the immunosuppressive agent is an anti-inflammatoryagent.
 51. The method of claim 50, wherein the anti-inflammatory agentis a cyclooxygenase inhibitor, a 5-lipoxygenase inhibitor, or aleukotriene receptor antagonist.
 52. The method of claim 1, wherein thefirst antibody is conjugated to a cytotoxic agent.
 53. The method ofclaim 52, wherein the cytotoxic agent is selected from the groupconsisting of an enediyne, a lexitropsin, a duocarmycin, a taxane, apuromycin, a dolastatin, a maytansinoid, a DNA minor groove bindingagent, a DNA minor groove alkylating agent, and a vinca alkaloid. 54.The method of claim 52, wherein the cytotoxic agent is paclitaxel,docetaxel. CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin,cyanomorpholino-doxorubicin, dolastatin-10, echinomycin, combretastatin,calicheamicin, maytansine, DM-1, auristatin E, AEB. AEVB, AEFP, MMAE, ornetropsin.
 55. The method of claim 52, wherein the cytotoxic agent is ananti-tubulin agent.
 56. The method of claim 55, wherein the cytotoxicagent is a vinca alkaloid, a podophyllotoxin, a taxane, a baccatinderivative, a cryptophysin, a maytansinoid, a combretastatin, or adolastatin.
 57. The method of claim 55, wherein the cytotoxic agent isvincristine, vinblastine, vindesine, vinorelbine, VP-16, camptothecin,paclitaxel, docetaxel, epithilone A, epithilone B, nocodazole,colchicine, colcimid, estramustine, cemadotin, discodermolide,maytansine, DM-1, auristatin E, AEB, AEVB, AEFP, MMAE, or eleutherobin.58. The method of claim 52, wherein the cytotoxic agent is MMAE.
 59. Themethod of claim 52, wherein the cytotoxic agent is AEFP.
 60. The methodof claim 52, wherein the first antibody is conjugated to the cytotoxicagent via a peptide linker.
 61. The method of claim 52, wherein thefirst antibody is conjugated to the cytotoxic agent via a val-cit linkeror a phe-lys linker.
 62. The method of claim 52, wherein the firstantibody is conjugated to the cytotoxic agent via a hydrazone-linker, ora disulfide-linker.
 63. The method of claim 52, wherein the conjugate iscAC10-val-cit-MMAE.
 64. The method of claim 52, wherein the conjugate iscAC10-val-cit-AEFP.
 65. The method of claim 52, wherein the firstantibody is conjugated to the cytotoxic agent via a linker that ishydrolyzable at a pH of less than 5.5.
 66. The method of claim 65,wherein the linker is hydrolyzable at a pH of less than 5.0.
 67. Themethod of claim 65, wherein the linker is a hydrazone linker or adisulfide linker.
 68. The method of claim 52, wherein the first antibodyis conjugated to the cytotoxic agent via a linker, wherein the linker iscleavable by a protease.
 69. The method of claim 52, wherein the firstantibody is conjugated to the cytotoxic agent via a peptide linker, andwherein the linker is cleavable by a protease.
 70. The method of claim68, wherein the protease is a membrane-associated protease.
 71. Themethod of claim 68, wherein the protease is an intracellular protease.72. The method of claim 68, wherein the protease is an endosomalprotease.
 73. The method of claim 68, wherein the protease is alysosomal protease.
 74. The method of claim 52, wherein the firstantibody is a monoclonal antibody, a chimeric antibody, a humanantibody, a humanized antibody, a glycosylated antibody, a multispecificantibody, a single-chain antibody, a Fab fragment, a F(ab′) fragment, aF(ab′)₂ fragment, a Fd, a single-chain Fv, a disulfide-linked Fv, afragment comprising a V_(L) domain, a polypeptide that bindsspecifically to CD30, or a fragment comprising a V_(H) domain.
 75. Themethod of claim 1, wherein the first antibody is conjugated to aimmunosuppressive agent.
 76. The method of claim 75, wherein theimmunosuppressive agent is gancyclovir, etanercept, cyclosporine,tacrolimus, or rapamycin.
 77. The method of claim 75, wherein theimmunosuppressive agent is an alkylating agent.
 78. The method of claim77, wherein the alkylating agent is cyclophosphamide.
 79. The method ofclaim 75, wherein the immunosuppressive agent is an antimetabolite. 80.The method of claim 79, wherein the antimetabolite is a purineantagonist.
 81. The method of claim 80, wherein the purine antagonist isazathioprine, or mycophenolate mofetil.
 82. The method of claim 79,wherein the antimetabolite is a dihydrofolate reductase inhibitor. 83.The method of claim 82, wherein the dihydrofolate reductase inhibitor ismethotrexate.
 84. The method of claim 75, wherein the immunosuppressiveagent is a glucocorticoid.
 85. The method of claim 84, wherein theglucocorticoid is cortisol or aldosterone.
 86. The method of claim 75,wherein the immunosuppressive agent is a glucocorticoid analogue. 87.The method of claim 86, wherein the glucocorticoid analogue isprednisone or dexamethasone.
 88. The method of claim 75, wherein theimmunosuppressive agent is an anti-inflammatory agent.
 89. The method ofclaim 88, wherein the anti-inflammatory agent is a cyclooxygenaseinhibitor, a 5-lipoxygenase inhibitor, or a leukotriene receptorantagonist.
 90. The method of claim 1, wherein the immunologicaldisorder is a Th₂-lymphocyte related disorder.
 91. The method of claim90, wherein the immunological disorder is atopic dermatitis, systemiclupus erythematosus, atopic asthma, rhinoconjunctivitis, allergicrhinitis, Omenn's syndrome, systemic sclerosis, or chronic graft versushost disease.
 92. The method of claim 1, wherein the immunologicaldisorder is a Th₁ lymphocyte-related disorder.
 93. The method of claim92, wherein the immunological disorder is rheumatoid arthritis, multiplesclerosis, psoriasis, Sjorgren's syndrome, Hashimoto's thyroiditis,Grave's disease, primary biliary cirrhosis, Wegener's granulomatosis,tuberculosis, or acute graft versus host disease.
 94. The method ofclaim 1, wherein the immunological disorder is due to viral infection.95. The method of claim 94, wherein the viral infection involves theEpstein-Barr virus, human immunodeficiency virus, human T leukemiavirus, hepatitis B virus, or measles virus.
 96. The method of claim 1,wherein the immunological disorder is an activated B lymphocyte-relateddisorder.
 97. A method for the treatment of an immunological disorder ina subject, wherein the immunological disorder is not cancer, comprisingadministering to the subject, in an amount effective for said treatment,a pharmaceutical composition comprising (a) a first antibody that (i)immunospecifically binds CD30 and (ii) induces CD30 signaling in alymphocyte; and (b) a pharmaceutically acceptable carrier.
 98. Themethod of claim 97, wherein the first antibody is human, humanized orchimeric.
 99. The method of claim 97, wherein the first antibody ismultivalent.
 100. The method of claim 97, wherein the first antibodycompetes for binding to CD30 with monoclonal antibodies AC10 or HeFi-1.101. The method of claim 97, wherein the first antibody is capable ofinducing CD30 signaling in the absence of cells other than thelymphocyte.
 102. The method of claim 97, wherein the first antibody iscapable of inducing CD30 signaling as a monospecific antibody.
 103. Themethod of claim 97, further comprising administering a second antibody.104. The method of claim 97, wherein the second antibody recognizes asecond receptor or receptor complex expressed on activated lymphocytes.105. The method of claim 104, wherein the receptor or the receptorcomplex comprises an immunoglobulin gene superfamily member, a TNFreceptor superfamily member, an integrin, a cytokine receptor, achemokine receptor, a major histocompatibility protein, a lectin, or acomplement control protein.
 106. The method of claim 105, wherein theimmunoglobulin superfamily member is CD2, CD3, CD4, CD8, CD19, CD22,CD28, CD79, CD90, CD152/CTLA-4, PD-1, or ICOS.
 107. The method of claim105, wherein the TNF receptor superfamily member is CD27, CD40,CD95/Fas, CD134/OX40, CD137/4-1BB, TNF-R1, TNFR-2, RANK, TACI, BCMA,osteoprotegerin, Apo2/TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4, or APO-3.108. The method of claim 105, wherein the integrin is CD11a, CD11b,CD11c, CD18, CD29, CD41, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f,CD103, or CD104.
 109. The method of claim 105, wherein the lectin isC-type, S-type, or I-type lectin.
 110. The method of claim 97, whereinthe first antibody is a bispecific antibody.
 111. The method of claim110, wherein the wherein the bispecific antibody binds to CD30 and asecond receptor or receptor complex expressed on activated lymphocytes.112. The method of claim 111, wherein the receptor or receptor complexcomprises an immunoglobulin gene superfamily member, a TNF receptorsuperfamily member, an integrin, a cytokine receptor, a chemokinereceptor, a major histocompatibility protein, a lectin, or a complementcontrol protein.
 113. The method of claim 112, wherein theimmunoglobulin superfamily member is CD2, CD3, CD4, CD8, CD19, CD22,CD28, CD79, CD90, CD152/CTLA4, PD-1, or ICOS.
 114. The method of claim112, wherein the TNF receptor superfamily member is CD27, CD40,CD95/Fas, CD134/OX40, CD137/4-1BB, TNF-R1, TNFR-2, RANK, TACI, BCMA,osteoprotegerin, Apo2/TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4, or APO-3.115. The method of claim 112, wherein the integrin is CD11a, CD11b,CD11c, CD18, CD29, CD41, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f,CD103, or CD104.
 116. The method of claim 112, wherein the lectin isC-type, S-type, or I-type lectin.
 117. The method of claim 97, furthercomprising administering a ligand that binds to a receptor or receptorcomplex expressed on activated lymphocytes.
 118. The method of claim117, wherein the receptor or receptor complex comprises animmunoglobulin gene superfamily member, a TNF receptor superfamilymember, an integrin, a cytokine receptor, a chemokine receptor, a majorhistocompatibility protein, a lectin, or a complement control protein.119. The method of claim 118, wherein the immunoglobulin superfamilymember is CD2, CD3, CD4, CD8, CD19, CD22, CD28, CD79, CD90,CD152/CTLA-4, PD-1, or ICOS.
 120. The method of claim 118, wherein theTNF receptor superfamily member is CD27, CD40, CD95/Fas, CD134/OX40,CD137/4-1BB, TNF-R1, TNFR-2, RANK, TACI, BCMA, osteoprotegerin,Apo2/TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4, or APO-3.
 121. The methodof claim 118, wherein the integrin is CD11a, CD11b, CD11c, CD18, CD29,CD41, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD103, or CD104. 122.The method of claim 118, wherein the lectin is C-type, S-type, or I-typelectin.
 123. The method of claim 97, wherein the first antibody is afusion protein comprising the amino acid sequence of a second proteinthat is not an antibody.
 124. The method of claim 123, wherein thesecond protein confers multivalent binding properties to the firstantibody.
 125. The method of claim 97, 103, 104, 110, or 111, furthercomprising administering an immunosuppressive agent.
 126. The method ofclaim 125, wherein the immunosuppressive agent is gancyclovir,etanercept, cyclosporine, tacrolimus, or rapamycin.
 127. The method ofclaim 125, wherein the immunosuppressive agent is an alkylating agent.128. The method of claim 127, wherein the alkylating agent iscyclophosphamide.
 129. The method of claim 125, wherein theimmunosuppressive agent is an antimetabolite.
 130. The method of claim129, wherein the antimetabolite is a purine antagonist.
 131. The methodof claim 130, wherein the purine antagonist is azathioprine, ormycophenolate mofetil.
 132. The method of claim 129, wherein theantimetabolite is a dihydrofolate reductase inhibitor.
 133. The methodof claim 132, wherein the dihydrofolate reductase inhibitor ismethotrexate.
 134. The method of claim 125, wherein theimmunosuppressive agent is a glucocorticoid.
 135. The method of claim134, wherein the glucocorticoid is cortisol or aldosterone.
 136. Themethod of claim 125, wherein the immunosuppressive agent is aglucocorticoid analogue.
 137. The method of claim 136, wherein theglucocorticoid analogue is prednisone or dexamethasone.
 138. The methodof claim 125, wherein the immunosuppressive agent is ananti-inflammatory agent.
 139. The method of claim 138, wherein theanti-inflammatory agent is a cyclooxygenase inhibitor, a 5-lipoxygenaseinhibitor, or a leukotriene receptor antagonist.
 140. The method ofclaim 97, wherein the first antibody is conjugated to a cytotoxic agent.141. The method of claim 140, wherein the cytotoxic agent is selectedfrom the group consisting of an enediyne, a lexitropsin, a duocarmycin,a taxane, a puromycin, a dolastatin, a maytansinoid, a DNA minor groovebinding agent, a DNA minor groove alkylating agent, and a vincaalkaloid.
 142. The method of claim 140, wherein the cytotoxic agent ispaclitaxel, docetaxel, CC-1065, SN-38, topotecan,morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin,dolastatin-10, echinomycin, combretastatin, calicheamicin, maytansine,DM-1, auristatin E, AEB, AEVB, AEFP, MMAE, or netropsin.
 143. The methodof claim 140, wherein the cytotoxic agent is an anti-tubulin agent. 144.The method of claim 143, wherein the cytotoxic agent is a vincaalkaloid, a podophyllotoxin, a taxane, a baccatin derivative, acryptophysin, a maytansinoid, a combretastatin, or a dolastatin. 145.The method of claim 143, wherein the cytotoxic agent is vincristine,vinblastine, vindesine, vinorelbine, VP-16, camptothecin, paclitaxel,docetaxel, epithilone A, epithilone B, nocodazole, colchicine, colcimid,estramustine, cemadotin, discodermolide, maytansine, DM-1, auristatin E,AEB, AEVB, AEFP, MMAE, or eleutherobin.
 146. The method of claim 140,wherein the cytotoxic agent is MMAE.
 147. The method of claim 140,wherein the cytotoxic agent is AEFP.
 148. The method of claim 140,wherein the first antibody is conjugated to the cytotoxic agent via apeptide linker.
 149. The method of claim 140, wherein the first antibodyis conjugated to the cytotoxic agent via a val-cit linker or a phe-lyslinker.
 150. The method of claim 140, wherein the first antibody isconjugated to the cytotoxic agent via a hydrazone-linker, or adisulfide-linker.
 151. The method of claim 140, wherein the firstantibody is conjugated to the cytotoxic agent via a linker that ishydrolyzable at a pH of less than 5.5.
 152. The method of claim 151,wherein the linker is hydrolyzable at a pH of less than 5.0.
 153. Themethod of claim 151, wherein the linker is a hydrazone linker or adisulfide linker.
 154. The method of claim 140, wherein the firstantibody is conjugated to the cytotoxic agent via a linker, wherein thelinker is cleavable by a protease.
 155. The method of claim 140, whereinthe first antibody is conjugated to the cytotoxic agent via a peptidelinker, and wherein the linker is cleavable by a protease.
 156. Themethod of claim 154, wherein the protease is a membrane-associatedprotease.
 157. The method of claim 154, wherein the protease is anintracellular protease.
 158. The method of claim 154, wherein theprotease is an endosomal protease.
 159. The method of claim 154, whereinthe protease is a lysosomal protease.
 160. The method of claim 140,wherein the first antibody is a monoclonal antibody, a chimericantibody, a human antibody, a humanized antibody, a glycosylatedantibody, a multispecific antibody, a single-chain antibody, a Fabfragment, a F(ab′) fragment, a F(ab′)₂ fragment, a Fd, a single-chainFv, a disulfide-linked Fv, a fragment comprising a V_(L) domain, apolypeptide that binds specifically to CD30, or a fragment comprising aV_(H) domain.
 161. The method of claim 97, wherein the first antibody isconjugated to a immunosuppressive agent.
 162. The method of claim 161,wherein the immunosuppressive agent is gancyclovir, etanercept,cyclosporine, tacrolimus, or rapamycin.
 163. The method of claim 161wherein the immunosuppressive agent is an alkylating agent.
 164. Themethod of claim 163, wherein the alkylating agent is cyclophosphamide.165. The method of claim 161, wherein the immunosuppressive agent is anantimetabolite.
 166. The method of claim 165, wherein the antimetaboliteis a purine antagonist.
 167. The method of claim 166, wherein the purineantagonist is azathioprine, or mycophenolate mofetil.
 168. The method ofclaim 165, wherein the antimetabolite is a dihydrofolate reductaseinhibitor.
 169. The method of claim 168, wherein the dihydrofolatereductase inhibitor is methotrexate.
 170. The method of claim 161,wherein the immunosuppressive agent is a glucocorticoid.
 171. The methodof claim 170, wherein the glucocorticoid is cortisol or aldosterone.172. The method of claim 161, wherein the immunosuppressive agent is aglucocorticoid analogue.
 173. The method of claim 172, wherein theglucocorticoid analogue is prednisone or dexamethasone.
 174. The methodof claim 161, wherein the immunosuppressive agent is ananti-inflammatory agent.
 175. The method of claim 174, wherein theanti-inflammatory; agent is a cyclooxygenase inhibitor, a 5-lipoxygenaseinhibitor, or a leukotriene receptor antagonist.
 176. The method ofclaim 97, wherein the immunological disorder is a Th₂-lymphocyte relateddisorder.
 177. The method of claim 176, wherein the immunologicaldisorder is atopic dermatitis, systemic lupus erythematosus, atopicasthma, rhinoconjunctivitis, allergic rhinitis, Omenn's syndrome,systemic sclerosis, or chronic graft versus host disease.
 178. Themethod of claim 97, wherein the immunological disorder is a Th₁lymphocyte-related disorder.
 179. The method of claim 178, wherein theimmunological disorder is rheumatoid arthritis, multiple sclerosis,psoriasis, Sjorgren's syndrome, Hashimoto's thyroiditis, Grave'sdisease, primary biliary cirrhosis, Wegener's granulomatosis,tuberculosis, or acute graft versus host disease.
 180. The method ofclaim 97, wherein the immunological disorder is due to viral infection.181. The method of claim 180, wherein the viral infection involves theEpstein-Barr virus, human immunodeficiency virus, human T leukemiavirus, hepatitis B virus, or measles virus.
 182. The method of claim 97,wherein the immunological disorder is an activated B lymphocyte-relateddisorder.
 183. A method for the treatment of an immunological disorderin a subject, wherein the immunological disorder is not cancer,comprising administering to the subject, in an amount effective for saidtreatment, a pharmaceutical composition comprising (a) an antibody that(i) immunospecifically binds CD30 and (ii) competes for binding to CD30with monoclonal antibody AC10 or HeFi-1; and (b) a pharmaceuticallyacceptable carrier.
 184. A method for the treatment of an immunologicaldisorder in a subject, wherein the immunological disorder is not cancer,comprising administering to the subject, in an amount effective for saidtreatment, a pharmaceutical composition comprising (a) an antibody that(i) immunospecifically binds CD30 and (ii) comprises SEQ ID NO:2; and(b) a pharmaceutically acceptable carrier.
 185. A method for thetreatment of an immunological disorder in a subject, wherein theimmunological disorder is not cancer, comprising administering to thesubject, in an amount effective for said treatment, a pharmaceuticalcomposition comprising (a) an antibody that (i) immunospecifically bindsCD30 and (ii) comprises one, two or all of: SEQ ID NO:4, SEQ ID NO:6 andSEQ ID NO:8; and (b) a pharmaceutically acceptable carrier.
 186. Amethod for the treatment of an immunological disorder in a subject,wherein the immunological disorder is not cancer, comprisingadministering to the subject, in an amount effective for said treatment,a pharmaceutical composition comprising (a) an antibody that (i)immunospecifically binds CD30 and (ii) comprises SEQ ID NO: 18; and (b)a pharmaceutically acceptable carrier.
 187. A method for the treatmentof an immunological disorder in a subject, wherein the immunologicaldisorder is not cancer, comprising administering to the subject, in anamount effective for said treatment, a pharmaceutical compositioncomprising (a) an antibody that (i) immunospecifically binds CD30 and(ii) comprises one, two or all of: SEQ ID NO:20, SEQ ID NO:22 and SEQ IDNO:24; and (b) a pharmaceutically acceptable carrier.
 188. A method forthe treatment of an immunological disorder in a subject, wherein theimmunological disorder is not cancer, comprising administering to thesubject, in an amount effective for said treatment, a pharmaceuticalcomposition comprising (a) an antibody that (i) immunospecifically bindsCD30 and (ii) competes for binding to CD30 with monoclonal antibody AC10or HeFi-1, wherein said antibody is conjugated to a cytotoxic agent; and(b) a pharmaceutically acceptable carrier.
 189. The method of claim 188,wherein the cytotoxic agent is selected from the group consisting of anenediyne, a lexitropsin, a duocarmycin, a taxane, a puromycin, adolastatin, a maytansinoid, a DNA minor groove binding agent, a DNAminor groove alkylating agent, and a vinca alkaloid.
 190. The method ofclaim 188, wherein the cytotoxic agent is paclitaxel, docetaxel,CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin,cyanomorpholino-doxorubicin, dolastatin-10, echinomycin, combretastatin,calicheamicin, maytansine, DM-1, auristatin E, AEB, AEVB, AEFP, MMAE, ornetropsin.
 191. The method of claim 188, wherein the cytotoxic agent isan anti-tubulin agent.
 192. The method of claim 191, wherein thecytotoxic agent is a vinca alkaloid, a podophyllotoxin, a taxane, abaccatin derivative, a cryptophysin, a maytansinoid, a combretastatin,or a dolastatin.
 193. The method of claim 191, wherein the cytotoxicagent is vincristine, vinblastine, vindesine, vinorelbine, VP-16,camptothecin, paclitaxel, docetaxel, epithilone A, epithilone B,nocodazole, colchicine, colcimid, estramustine, cemadotin,discodermolide, maytansine, DM-1, auristatin E, AEB, AEVB, AEFP, MMAE,or eleutherobin.
 194. The method of claim 188, wherein the cytotoxicagent is MMAE.
 195. The method of claim 188, wherein the cytotoxic agentis AEFP.
 196. The method of claim 188, wherein the first antibody isconjugated to the cytotoxic agent via a peptide linker.
 197. The methodof claim 188, wherein the first antibody is conjugated to the cytotoxicagent via a val-cit linker or a phe-lys linker.
 198. The method of claim18S, wherein the first antibody is conjugated to the cytotoxic agent viaa hydrazone-linker, or a disulfide-linker.
 199. The method of claim 188,wherein the first antibody is conjugated to the cytotoxic agent via alinker that is hydrolyzable at a pH of less than 5.5.
 200. The method ofclaim 199, wherein the linker is hydrolyzable at a pH of less than 5.0.201. The method of claim 199, wherein the linker is a hydrazone linkeror a disulfide linker.
 202. The method of claim 18S, wherein the firstantibody is conjugated to the cytotoxic agent via a linker, wherein thelinker is cleavable by a protease.
 203. The method of claim 188 whereinthe first antibody is conjugated to the cytotoxic agent via a peptidelinker, and wherein the linker is cleavable by a protease.
 204. Themethod of claim 202, wherein the protease is a membrane-associatedprotease.
 205. The method of claim 202, wherein the protease is anintracellular protease.
 206. The method of claim 202, wherein theprotease is an endosomal protease.
 207. The method of claim 202, whereinthe protease is a lysosomal protease.
 208. The method of claim 188,wherein the first antibody is a monoclonal antibody, a chimericantibody, a human antibody, a humanized antibody, a glycosylatedantibody, a multi specific antibody, a single-chain antibody, a Fabfragment, a F(ab′) fragment, a F(ab′)₂ fragment, a Fd, a single-chainFv, a disulfide-linked Fv, a fragment comprising a VL domain, apolypeptide that binds specifically to CD30, or a fragment comprising aVH domain.
 209. A method for the treatment of an immunological disorderin a subject, wherein the immunological disorder is not cancer,comprising administering to the subject, in an amount effective for saidtreatment, a pharmaceutical composition comprising (a)cAC10-val-cit-MMAE; and (b) a pharmaceutically acceptable carrier. 210.A method for the treatment of an immunological disorder in a subject,wherein the immunological disorder is not cancer, comprisingadministering to the subject, in an amount effective for said treatment,a pharmaceutical composition comprising (a) cAC10-val-cit-AEFP; and (b)a pharmaceutically acceptable carrier.